Correction: Integrated single-cell and bulk gene expression and ATAC-seq reveals heterogeneity and early changes in pathways associated with resistance to cetuximab in HNSCC-sensitive cell lines

Abstract Multiple myeloma (MM) is characterized by significant genetic diversity at subclonal levels that likely plays a defining role in the heterogeneity of tumor progression, clinical aggressiveness and drug sensitivity. Such heterogeneity is a driving factor in the evolution of MM, from founder clones through outgrowth of subclonal fractions. DNA Sequencing studies on MM samples have indeed demonstrated such heterogeneity in subclonal architecture at diagnosis based on recurrent mutations in pathologically relevant genes that may ultimately to lead to relapse. However, no study so far has reported a predictive gene expression signature that can identify, distinguish and quantify drug sensitive and drug-resistant subpopulations within a bulk population of myeloma cells. In recent years, our laboratory has successfully developed a gene expression profile (GEP)-based signature that could not only distinguish drug response of MM cell lines, but also was effective in stratifying patient outcomes when applied to GEP profiles from MM clinical trials using proteasome inhibitors (PI) as chemotherapeutic agents. Further, we noted myeloma cell lines that responded to the drug often contained residual sub-population of cells that did not respond, and likely were selectively propagated during drug treatment in vitro, and in patients. In this study, we performed targeted qRT-PCR analysis of single cells using a gene panel that included PI sensitivity genes and gene signatures that could discriminate between low and high-risk myeloma followed by intensive bioinformatics and statistical analysis for the classification and prediction of PI response in individual cells within bulk multiple myeloma tumors. Fluidigm's C1 Single-Cell Auto Prep System was used to perform automated single-cell capture, processing and cDNA synthesis on 576 pre-treatment cells from 12 cell lines representing a wide range of PI-sensitivity and 370 cells from 7 patient samples undergoing PI treatment followed by targeted gene expression profiling of single cells using automated, high-throughput on-chip qRT-PCR analysis using 96.96 Dynamic Array IFCs on the BioMark HD System. Probability of resistance for each individual cell was predicted using a pipeline that employed the machine learning methods Random Forest, Support Vector Machine (radial and sigmoidal), LASSO and kNN (k Nearest Neighbor) for making single-cell GEP data-driven predictions/ decisions. The weighted probabilities from each of the algorithms were used to quantify resistance of each individual cell and plotted using Ensemble forecasting algorithm. Using our drug response GEP signature at the single cell level, we could successfully identify distinct subpopulations of tumor cells that were predicted to be sensitive or resistant to PIs. Subsequently, we developed a R Statistical analysis package (http://cran.r-project.org), SCATTome (Single Cell Analysis of Targeted Transcriptome), that can restructure data obtained from Fluidigm qPCR analysis run, filter missing data, perform scaling of filtered data, build classification models and successfully predict drug response of individual cells and classify each cell's probability of response based on the targeted transcriptome. We will present the program output as graphical displays of single cell response probabilities. This package provides a novel classification method that has the potential to predict subclonal response to a variety of therapeutic agents. Disclosures Kumar: Skyline: Consultancy, Honoraria; BMS: Consultancy; Onyx: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Novartis: Research Funding; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding.

Download Full-text

Comprehensive RNA analysis of CSF reveals a role for CEACAM6 in lung cancer leptomeningeal metastases

npj Precision Oncology ◽

10.1038/s41698-021-00228-6 ◽

2021 ◽

Vol 5 (1) ◽

Author(s):

Yingmei Li ◽

Dina Polyak ◽

Layton Lamsam ◽

Ian David Connolly ◽

Eli Johnson ◽

...

Keyword(s):

Gene Expression ◽

Lung Cancer ◽

Single Cell ◽

Cell Lines ◽

Expression Profiles ◽

Leptomeningeal Metastases ◽

Detection Sensitivity ◽

Nsclc Cell ◽

Rna Seq ◽

The Brain

AbstractNon-small cell lung cancer (NSCLC) metastatic to the brain leptomeninges is rapidly fatal, cannot be biopsied, and cancer cells in the cerebrospinal fluid (CSF) are few; therefore, available tissue samples to develop effective treatments are severely limited. This study aimed to converge single-cell RNA-seq and cell-free RNA (cfRNA) analyses to both diagnose NSCLC leptomeningeal metastases (LM), and to use gene expression profiles to understand progression mechanisms of NSCLC in the brain leptomeninges. NSCLC patients with suspected LM underwent withdrawal of CSF via lumbar puncture. Four cytology-positive CSF samples underwent single-cell capture (n = 197 cells) by microfluidic chip. Using robust principal component analyses, NSCLC LM cell gene expression was compared to immune cells. Massively parallel qPCR (9216 simultaneous reactions) on human CSF cfRNA samples compared the relative gene expression of patients with NSCLC LM (n = 14) to non-tumor controls (n = 7). The NSCLC-associated gene, CEACAM6, underwent in vitro validation in NSCLC cell lines for involvement in pathologic behaviors characteristic of LM. NSCLC LM gene expression revealed by single-cell RNA-seq was also reflected in CSF cfRNA of cytology-positive patients. Tumor-associated cfRNA (e.g., CEACAM6, MUC1) was present in NSCLC LM patients’ CSF, but not in controls (CEACAM6 detection sensitivity 88.24% and specificity 100%). Cell migration in NSCLC cell lines was directly proportional to CEACAM6 expression, suggesting a role in disease progression. NSCLC-associated cfRNA is detectable in the CSF of patients with LM, and corresponds to the gene expression profile of NSCLC LM cells. CEACAM6 contributes significantly to NSCLC migration, a hallmark of LM pathophysiology.

Download Full-text

Molecular and Functional Effects of the Novel MEK Inhibitor PD0325901 in Preclinical Models of Human Leukemias.

Blood ◽

10.1182/blood.v108.11.254.254 ◽

2006 ◽

Vol 108 (11) ◽

pp. 254-254

Author(s):

Michele Milella ◽

Maria Rosaria Ricciardi ◽

Chiara Gregorj ◽

Fabiana De Cave ◽

Steven L. Abrams ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Proliferation ◽

Cell Line ◽

Cell Lines ◽

Therapeutic Target ◽

Hematological Malignancies ◽

Mek Inhibitor ◽

Sensitive Cell ◽

Fold Reduction

Abstract The Raf/MEK/ERK signaling module plays a pivotal role in the regulation of cell proliferation, survival, and differentiation. Our group, among others, has recently demonstrated that this pathway is frequently dysregulated in hematological malignancies and may constitute an attractive therapeutic target, particularly in AML. Here we investigated the effects of PD0325901, a novel MEK inhibitor, on phospho-protein expression, gene expression profiles, cell proliferation, and apoptosis in cell line models of AML, ALL, multiple myeloma (MM), ex vivo-cultured primary AML blasts, and oncogene-transformed hematopoietic cells. AML cell lines (OCI-AML2, OCI-AML3, HL-60) were strikingly sensitive to PD0325901 (IC50: 5–19 nM), NB4 (APL) and U266 (MM) showed intermediate sensitivity (IC50: 822 and 724 nM), while all the lymphoid cell lines tested and the myeloid cell lines U937 and KG1 were resistant (IC50 > 1000 nM). Cell growth inhibition was due to inhibition of cell cycle progression and induction of apoptosis. A statistically significant reduction in the proportion of S-phase cells (p=0.01) and increase in the percentage of apoptotic cells (p=0.019) was also observed in 18 primary AML samples in response to 100 nM PD0325901. Analysis of the correlation between sensitivity/resistance to PD0325901 and Ras/Raf mutation status is currently ongoing. PD0325901 effects were also examined in a panel of IL-3-dependent murine myeloid FDC-P1 cell lines transformed to grow in response to 11 different oncogenes in the absence of IL-3. Fms-, Ras-, Raf-1-, B-Raf-, MEK1-, IGF-1R-, and STAT5a-transformed FDC-P1 cells were very sensitive to PD0325901 (IC50: ~ 1 nM), while A-Raf-, BCR-ABL-, EGFR- or Src-transformed cells were 10 to 100 fold less sensitive (IC50: 10 to 100 nM); the parental, IL-3 dependent FDC-P1 cell line had an IC50 > 1000 nM. Analysis of the phosphorylation levels of 18 different target proteins after treatment with 10 nM PD0325901 showed a 5- to 8-fold reduction in ERK-1/2, observed only in sensitive cell lines, and a 2-fold reduction in JNK and STAT3 phosphorylation. PD0325901 (10 nM) treatment also profoundly altered the gene expression profile of the sensitive cell line OCI-AML3: 96 genes were modulated after 24 h (37 up- and 59 down-regulated), most of which involved in cell cycle regulation. Changes in cyclin D1 and D3, cyclin E, and cdc 25A were also validated at the protein level. Overall, PD0325901 shows potent growth-inhibitory and pro-apoptotic activity, indicating that MEK may be an appropriate therapeutic target in an array of different hematological malignancies. Further preclinical/clinical development of this compound is warranted, particularly in myeloid leukemias.

Download Full-text

Single-Cell Transcriptomics Identifies Intra-Tumor Heterogeneity in Human Myeloma Cell Lines

Blood ◽

10.1182/blood.v124.21.3385.3385 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3385-3385 ◽

Cited By ~ 3

Author(s):

Amit Kumar Mitra ◽

Holly Stessman ◽

Michael A. Linden ◽

Brian Van Ness

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Lines ◽

Tumor Heterogeneity ◽

Myeloma Cell ◽

Gene Model ◽

Color Flow ◽

Myeloma Cells ◽

Genetic Signature ◽

Human Myeloma Cell

Abstract Multiple myeloma (MM) is a plasma cell neoplasm with significant complexity and heterogeneity. Proteasome inhibitors (PI) including bortezomib (Velcade/Bz), carfilzomib (Kyprolis/Cz) and Ixazomib are effective chemotherapeutic agents in the treatment of MM, used alone or in combination with other anti-cancer agents. However, in spite of the recent improvements in treatment strategies, MM still remains a difficult disease to cure with median survival rate of around 7 years. In a recently published study, we have shown that the heterogeneity in response to proteasome inhibitor (PI)-based treatment in MM is governed by underlying molecular characteristics of the subclones within tumor population (Stessman et al. 2013). We confirmed the presence of residual resistant sub-population comprising up to 15% of the bulk Bz-sensitive cell population in drug-naïve MM tumors. We hypothesize that this pre-existing resistant sub-population may give rise to emerging resistance in course of treatment with PIs. In the current study, we used single cell transcriptomics analysis to identify tumor subclones within Human Myeloma Cell Lines (HMCLs) based on a 48-gene model of predictive genetic signature for baseline PI response. Automated single-cell capture and cDNA synthesis from cellular RNA were performed using Fluidigm’s C1TM Single-Cell Auto Prep System. The cDNA was then harvested and transferred to BioMark HD System for single-cell targeted high-throughput qPCR-based gene expression analysis of a 48 gene-panel using Fluidigm DELTAgene assays. Our 48-gene model combines our previously published 23 gene expression profiling (GEP) signature that could discriminate between sensitive and resistant responsiveness to Bz, and the Shaughnessy et al prognostic 17-gene GEP model along with control genes, including cell cycle genes, anti-apoptotic genes, proteasome subunit genes, house-keeping genes and internal negative controls. Based on the differential expression of these 48 genes used in the modeling, distinct subclonal populations were then identified using a combination of Fluidigm’s analysis software and the R Statistical analysis package. Further, a principal component analysis (PCA) score plot was generated as a two-dimensional grid to visualize the separate populations associated with resistant profiles. Finally, hierarchical clustering (HC) analysis was used to generate heat maps that group expression patterns associated with response. Our results demonstrated the presence of pre-existing subclones of cells within untreated myeloma cells with a characteristic genetic signature profile distinct from the pre-treatment overall (bulk) profile of myeloma cells. As an additional validation of subclonal architecture, we demonstrated the presence of subclones within HMCLs using multi-color flow cytometry. The results presented will help identify the presence and extent of intra-tumor heterogeneity in MM by single cell transcriptomics and may define residual pre-existing subclones resistant to PI therapies. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Development and Therapeutic Potential of an Anti-CD79b Antibody-Drug Conjugate, Anti-CD79b-Vc-MMAE, for the Treatment of Non-Hodgkin’s Lymphoma

Blood ◽

10.1182/blood.v112.11.2618.2618 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2618-2618

Author(s):

Andrew G. Polson ◽

Fiona L Bennett ◽

Yvonne Chen ◽

Mark Dennis ◽

Dan Eaton ◽

...

Keyword(s):

Gene Expression ◽

Cell Surface ◽

Cell Lines ◽

Surface Expression ◽

Hodgkin's Lymphoma ◽

Cell Surface Expression ◽

Sensitive Cell ◽

Large Panel ◽

Xenograft Models ◽

Antibody Drug

Abstract Antibody-drug conjugates (ADCs), antibodies linked to potent cytotoxic drugs via specialized chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. We have previously shown that ADCs targeted to CD79b are highly effective in xenograft models of non-Hodgkin’s lymphoma. Here we report the development of an ADC consisting of a humanized anti-CD79b antibody (hu-anti-CD79b) conjugated to monomethylauristatin E (MMAE) through engineered cysteines (THIOMABS) by a maleimidocaproyl-valinecitrulline-p-aminobenzyloxycarbonyl (MC-vcPAB) linker (hu-anti-CD79b-thioMAb-MC-vc-PAB-MMAE) that is designed to be cleaved by cathepsins. To determine the potential of this ADC, hu-anti-CD79b-thioMAb-MC-vc-PAB-MMAE (referred to as anti-CD79b-vcMMAE henceforth), as a therapeutic for NHL we interrogated its potency across a large panel of NHL cell lines. Strikingly, anti-CD79b-vcMMAE has very potent activity across a large panel of NHL cell lines in vitro with 68% (23 out of 34) cell lines having greater than 50% reduction in cell viability. Quantitative FACS across the cell line panel revealed that of the 11 insensitive cell lines, 9 had negligible surface CD79b, suggesting a threshold effect in that below a specific level of antigen on the cell surface resulted in cells being insensitive to anti-CD79b-vcMMAE. Within the sensitive cell lines, there was not a direct correlation per se with anti-CD79b-vcMMAE IC50 values and cell surface expression levels and this prompted us to investigate other potential molecular parameters. Gene expression profiling and gene set enrichment analysis revealed that genes predominantly involved in antigen processing and presentation and genes induced by IFN-gamma were significantly enriched in the less sensitive cell lines to CD79b-vcMMAE. Classifying our NHL cell lines as GCB or ABC subtypes by gene expression revealed that both ABC and GCB were responsive. In addition, the activity of anti-CD79b-vcMMAE was very potent in p53 mutant as well as p53 wild-type cell lines. Since the pre-clinical evidence suggests that anti-CD79b-vcMMAE will be a very promising drug candidate for NHL and we have established that cell surface expression is perhaps the best predictor of response, we wished to determine the prevalence of expression of CD79b on the cell surface of primary human lymphoma and CLL samples to estimate a patient population that may gain benefit. Strikingly, CD79b was detected in all cases of CLL, MZL, HCL, DLBCL, FL, and MCL. Furthermore, CD79b expression was detected in all cases that had relapsed from prior chemotherapy regimens, highlighting the clinical relevance of this target and potential therapeutic utility of anti-CD79b-vcMMAE. To assess the potential of anti-CD79b-vcMMAE in vivo we compared its efficacy to R-CHOP in three xenograft models of NHL. In all three models, a single dose of anti-CD79b-vcMMAE resulted in complete sustained tumor remission and was more effective than R-CHOP. These data suggest that anti-CD79b-vcMMAE could be broadly efficacious as a treatment for NHL.

Download Full-text

Down-Regulation of BMI-1 Is a New Marker of Sensitivity to Mdm2 Inhibition in B-Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v120.21.2522.2522 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2522-2522 ◽

Cited By ~ 1

Author(s):

Ilaria Iacobucci ◽

Daniela Erriquez ◽

Anna Ferrari ◽

Cristina Papayannidis ◽

Claudia Venturi ◽

...

Keyword(s):

Gene Expression ◽

Western Blot ◽

Cell Viability ◽

Cell Lines ◽

Leukemia Cell ◽

Annexin V ◽

Sensitive Cell ◽

Protein Levels ◽

Leukemia Cell Lines ◽

Reh Cells

Abstract Abstract 2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods: TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roche. Results: BCR-ABL1-positive (BV-173, SUPB-15) and negative (NALM19, REH) ALL cell lines were investigated for TP53 mutations and CDKN2A deletion. A p53 mutation (R181C) was identified in REH cells, whereas all the remaining cell lines resulted p53 wild-type but they were deleted in the locus containing CDKN2A. Leukemia cell lines were incubated with increasing concentrations of Nutlin-3a (0.005–2 μM) for 24, 48 and 72 hours (hrs). Mdm2 inhibition resulted in a dose and time-dependent cytotoxicity with IC50 at 24 hrs ranging from around 1.5 μM for BV-173 and SUPB-15 to 3.7 μM for NALM-19. By contrast, no significant changes in cell viability were observed in RHE p53-mutated cells after incubation with Mdm2i. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/PI analysis showed a significant increase in apoptosis after 24 hrs in sensitive cell lines and in primary leukemia blasts, whereas no apoptosis was observed in REH cells. To examine the possible mechanisms underlying Mdm2i-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased as soon as 24 hrs of incubation with Mdm2i. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis was next performed, comparing sensitive cell lines at 24 hrs of incubation with concentrations equal to the IC50 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. Additionally, experimental reduction of BMI1 protein levels results in apoptosis in tumor cells and increases susceptibility to cytotoxic agents and radiation therapy (Wu et al., 2011). Given the importance of BMI in the control of apoptosis, we investigated by western blot its pattern in treated and untreated cells, confirming a marked decrease as soon as 24 hrs of exposure to MDM2i both in leukemia cell lines and primary blast samples. Noteworthy, the BMI-1 levels remained constant in resistant cells. Conclusions: Inhibition of Mdm2 efficiently activates the p53 pathway promoting apoptosis. BMI-1 expression is markedly reduced in sensitive cells and it may be used as a biomarker of response. Evaluation of its expression before and after treatment in clinical settings will better gain insight into its role. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007 – 2009, INPDAP. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:BMS: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Download Full-text

Changes in gene expression profile in two multidrug resistant cell lines derived from a same drug sensitive cell line

Leukemia Research ◽

10.1016/j.leukres.2014.06.001 ◽

2014 ◽

Vol 38 (8) ◽

pp. 983-987 ◽

Cited By ~ 19

Author(s):

Miguel Angelo Martins Moreira ◽

Carolina Bagni ◽

Marcos Barcelos de Pinho ◽

Thaís Messias Mac-Cormick ◽

Mateus dos Santos Mota ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Gene Expression Profile ◽

Cell Lines ◽

Expression Profile ◽

Multidrug Resistant ◽

Resistant Cell ◽

Sensitive Cell ◽

Sensitive Cell Line

Download Full-text

Targeted transcript quantification in single disseminated cancer cells after whole transcriptome amplification

10.1101/616839 ◽

2019 ◽

Author(s):

Franziska C. Durst ◽

Ana Grujovic ◽

Iris Ganser ◽

Martin Hoffmann ◽

Peter Ugocsai ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Single Cell ◽

Cancer Cells ◽

Cell Lines ◽

Single Cells ◽

Qpcr Assay ◽

Transcript Expression ◽

Whole Transcriptome Amplification ◽

Whole Transcriptome

AbstractGene expression analysis of rare or heterogeneous cell populations such as disseminated cancer cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-amplification of WTA products, quantification of amplified cDNA yields and final qPCR quantification, to identify the most reliable and accurate workflow for quantitation of gene expression of the ERBB2 gene in DCCs. We found that absolute quantification outperforms relative quantification. We then validated the performance of our method on single cells of established breast cancer cell lines displaying distinct levels of HER2 protein. The different protein levels were faithfully reflected by transcript expression across the tested cell lines thereby proving the accuracy of our approach. Finally, we applied our method on patient-derived breast cancer DCCs. Here, we were able to measure ERBB2 expression levels in all HER2-positive DCCs. In addition, we could detect ERBB2 transcript expression even in HER2-negative DCCs, suggesting post-transcriptional mechanisms of HER2 loss in anti-HER2-treated DCCs. In summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of ERBB2 in DCCs.

Download Full-text

Single-Cell Gene Network Analysis and Transcriptional Landscape of MYCN-Amplified Neuroblastoma Cell Lines

Biomolecules ◽

10.3390/biom11020177 ◽

2021 ◽

Vol 11 (2) ◽

pp. 177

Author(s):

Daniele Mercatelli ◽

Nicola Balboni ◽

Alessandro Palma ◽

Emanuela Aleo ◽

Pietro Paolo Sanna ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Lines ◽

The United States ◽

Network Activity ◽

Cycle Phase ◽

Transcriptional Networks ◽

Pathway Enrichment Analysis ◽

Genomic Amplification ◽

Cellular Models

Neuroblastoma (NBL) is a pediatric cancer responsible for more than 15% of cancer deaths in children, with 800 new cases each year in the United States alone. Genomic amplification of the MYC oncogene family member MYCN characterizes a subset of high-risk pediatric neuroblastomas. Several cellular models have been implemented to study this disease over the years. Two of these, SK-N-BE-2-C (BE2C) and Kelly, are amongst the most used worldwide as models of MYCN-Amplified human NBL. Here, we provide a transcriptome-wide quantitative measurement of gene expression and transcriptional network activity in BE2C and Kelly cell lines at an unprecedented single-cell resolution. We obtained 1105 Kelly and 962 BE2C unsynchronized cells, with an average number of mapped reads/cell of roughly 38,000. The single-cell data recapitulate gene expression signatures previously generated from bulk RNA-Seq. We highlight low variance for commonly used housekeeping genes between different cells (ACTB, B2M and GAPDH), while showing higher than expected variance for metallothionein transcripts in Kelly cells. The high number of samples, despite the relatively low read coverage of single cells, allowed for robust pathway enrichment analysis and master regulator analysis (MRA), both of which highlight the more mesenchymal nature of BE2C cells as compared to Kelly cells, and the upregulation of TWIST1 and DNAJC1 transcriptional networks. We further defined master regulators at the single cell level and showed that MYCN is not constantly active or expressed within Kelly and BE2C cells, independently of cell cycle phase. The dataset, alongside a detailed and commented programming protocol to analyze it, is fully shared and reusable.

Download Full-text