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Multiscale profiling of enzyme activity in cancer [Bulk-seq]

ABSTRACT: Purpose: The goal of this study was to use enzyme activity as a proxy for profiling tumor progression and treatment response in an autochthonous mouse model of Alk-mutant non-small-cell lung cancer (NSCLC). The Eml4-Alk model was originally described in Maddalo et al., Nature 2014. This dataset describes bulk RNA-seq profiling of cells from Eml4-Alk lungs following administration of a protease-activatable probe, QZ1, and sorting single cells based on signal from this probe. Methods: Eml4-Alk mice were anesthetized, and QZ1-(PEG2K) was administered intravenously via tail vein injection. Lungs were excised two hours later, separated into lobes, tumors were microdissected, and single cell suspensions were prepared. FACS sorting was performed on a FACSAria II (BD). Flow cytometry data was analyzed by the FlowJo software (Treestar). At least 100,000 cells from each of the QZ1+ and QZ1- compartments were collected into RPMI-1640 + 2% heat-inactivated FBS and pelleted via centrifugation at 1800 rpm for 5 minutes. Cell pellets were lysed in Trizol (ThermoFisher), and RNA was extracted using RNEasy Mini Kits (Qiagen), and bulk RNA sequencing was performed. Libraries were prepared using the Clontech SMARTer Stranded Total RNAseq Kit (Clontech), precleaned, and sequenced using an Illumina NextSeq500 on an Illumina NextSeq flow cell. Results: Feature counting was performed on BAM files using the Rsubread package. Differential expression analysis on QZ1+ vs QZ1- cells was performed using the DESeq2 package in R. GSEA was performed using GenePattern, and results were visualized using the clusterProfiler R package. Conclusions: This study demonstrates a method for activity-based cell sorting that can be coupled to downstream phenotypic characterization via RNA-seq.

ORGANISM(S): Mus musculus

PROVIDER: GSE191071 | GEO | 2021/12/20

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study was to use enzyme activity as a proxy for profiling tumor progression and treatment response in an autochthonous mouse model of Alk-mutant non-small-cell lung cancer (NSCLC). The Eml4-Alk model was originally described in Maddalo et al., Nature 2014. Single-cell transcriptomic profiling of the Eml4-Alk NSCLC model was performed for unbiased discovery of the phenotypic landscape of Eml4-Alk and wild type mice. Methods: Single cell suspensions were prepared from excised lungs of Eml4-Alk and sex- and age-matched healthy, wild type C57/Bl6J mice. Suspensions were pooled from n=3 mice per condition, and then were enriched for cell viability and depleted for CD45+ cells. Single cells were processed using the 10X Genomics Single Cell 3’ platform using the Chromium Single Cell 3’ Library & Gel Bead Kit V2 kit (10X Genomics), per manufacturer’s protocol. Approximately 10,000 cells were loaded onto each channel and partitioned into Gel Beads in Emulsion (GEMs) in the 10x Chromium instrument. Following lysis of the captured cells, the released RNA was barcoded through reverse transcription in individual GEMs, and complementary DNA was generated and amplified. Libraries were constructed using a Single Cell 3’ Library and Gel Bead kit. The libraries were sequenced using an Illumina NovaSeq6000 sequencer on an Illumina NovaSeq SP flow cell. Results: Gene expression matrices were generated for each sample by the Cell Ranger (v.3.0.2) Pipeline coupled with mouse reference version GRCm38. The output filtered gene expression matrices were analyzed by Python software (v.3.9.0) with the scanpy package (v.1.7.2). For final analysis, genes expressed in at least three cells in the data and cells with > 200 genes detected were selected for further analyses, and low quality cells were removed based on number of total counts and percentage of mitochondrial genes expressed. Conclusions: This study provides the first (to the best of our knowledge) single-cell RNA-seq dataset of the Eml4-Alk autochthonous model of NSCLC.

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Multiscale profiling of enzyme activity in cancer [Bulk-seq]

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