Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Functional and Molecular Single-Cell Analyses Implicate PRDM14 in the Initiation of a Novel B-1 Cell Malignancy - Single Cell RNA sequencing experiments

ABSTRACT: Description: Molecular mechanisms underlying high-risk subtypes of leukemia remain elusive. PR domain-containing 14 (Prdm14) is a potent oncogene implicated in the initiation of many cancers, including leukemia. Here, we interrogate the heterogeneity of Prdm14-expressing cell types in a mouse model of T-cell acute lymphoblastic leukemia (T-ALL). We identified an abnormal B-1 cell-like population in pre-leukemic bone marrow. B-1 cells are a self-renewing population of unconventional B cells established during embryonic development. This dataset contains single-cell transcriptomic profiling of four samples. Cells from R26PR;Mx1-cre mice were sorted on GFP expression (“GFP+” and “GFP-” samples) or on a pro-B-1-like immunophenotype of CD11b- CD19+ CD127+ Sca-1+ (“Pre-leukemia” sample). As a control, immunophenotype-matched cells from R26PR mice were analyzed by scRNA-seq after sorting on CD11b- CD19+ CD127+ Sca-1+ (“Control” sample) (Supplemental Figure 2). Accordingly, we conducted scRNA-seq of four total samples: “GFP+”, “GFP-“, “Pre-leukemia”, and “Control”. The GFP+ and GFP- samples provide the cellular context to the bone marrow samples.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Mus musculus

SUBMITTER: Dustin Sokolowski

PROVIDER: E-MTAB-13160 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Genetic and non-genetic heterogeneity within cancer cell populations represents a major challenge to anti-cancer therapies. We currently lack robust methods to determine how pre-existing and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA-sequencing, we have developed TraCe-seq, a method that captures at clonal resolution the origin, fate, and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual EGFR inhibitors-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. To QC the TraCe-seq strategy, single-cell RNA-seq libraries were generated from a variety of human cancer cell lines transduced with the TraCe-seq library to validate the TraCe-seq strategy. Specifically, 5 different cell lines (PC9, MCF-10A, MDA-MB-231, NCI-H358, and NCI-H1373) were each transduced with a unique TraCe-seq barcode. The transduced cells were selected with puromycin only, dissociated to single cell suspensions, and then mixed together. The complex mixture of the 5 cell lines was profiled by 10X scRNA-seq. Furthermore, transduced NCI-H1373 cells were sorted by FACS to enrich for the top 50% of eGFP positive cells, and sorted cells were cultured briefly and used to construct scRNA-seq libraries and profiled by 10x scRNA-seq. To carry out the full TraCe-seq experiment, ~600 PC9 cells carrying unique TraCe-seq barcodes were expanded over 12 doublings to establish the barcoded population. A subset of the barcoded PC9 population was used to generate scRNA-seq libraries and profiled by 10x scRNA-seq prior to treatment to establish a baseline transcription profile for each barcoded clone. The rest of the cells were then treated for four days with 1 µM erlotinib, 1 µM GNE-069, or 1 µM GNE-104 respectively. scRNA-seq libraries were then generated form the treated cells and profiled by 10x scRNA-seq.

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Functional and Molecular Single-Cell Analyses Implicate PRDM14 in the Initiation of a Novel B-1 Cell Malignancy - Single Cell RNA sequencing experiments

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