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| Status |
Public on Apr 13, 2024 |
| Title |
Gene expression profiles at single cell level of lung epithelial cells from the Cracd WT and Cracd KO mice |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Tumor cell plasticity contributes to intratumoral heterogeneity and therapy resistance. Through cell plasticity, lung adenocarcinoma (LUAD) cells transform into neuroendocrinal (NE) cell-like tumor cells. However, the mechanisms of NE cell plasticity remain unclear. Depletion of CRACD tumor suppressor results in the de-repression of NE-related gene expression in the pulmonary epithelium. Similarly, Cracd KO augments intratumoral heterogeneity with upregulation of NE markers in LUAD animal models. Single-cell transcriptomic analysis showed that Cracd KO upregulates NE genes in AT2 pulmonary epithelial cells, accompanied by a cell de-differentiation state and activation of Sox2, Oct4, and Nanog pathways. The single-cell transcriptomes of LUAD patient tumors recapitulate that the distinct LUAD NE cell cluster is co-enriched with NE genes and SOX2, OCT4, and NANOG pathways. This study reveals an unexpected role of CRACD tumor suppressor in restricting cell plasticity inducing cell de-differentiation, which provides new insights into NE cell plasticity of LUAD.
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| Overall design |
Lungs were harvested from euthanized mice (Cracd WT or Cracd KO) after perfusing 10 ml of cold phosphate-buffered saline (PBS) into the right ventricle. The lung was digested in Leibovitz’s medium (Invitrogen) with 2 mg/mL Collagenase Type I (Worthington), 2 mg/mL Elastase (Worthington), and 2 mg/mL DNase I (Worthington) at 37 °C for 45 min. The tissue was triturated with a pipet every 15 min of digestion until homogenous. The digestion was stopped with FBS (Invitrogen) to a final concentration of 20%. The cells were filtered with a 70 μm cell strainer (Falcon) and spun down at 5,000 r/min for 1 min. The cell pellet was resuspended in red blood cell lysing buffer (Sigma) for 3 min, spun down at 5,000 r/min for 1 min, and washed with 1 mL ice-cold Leibovitz’s medium with 10% FBS. In single-cell RNA sequencing (scRNA-seq), digested lung cells were resuspended in 400 μl of buffer with 5 μl of anti-CD31-FITC (BD Biosciences, CA, USA), 5 μl of anti-CD45-APC (BD Biosciences), and 5 μl of anti-CD326 (EpCAM)-PE-Cy7 (BD Biosciences) and incubated for 30 min at 4 C. Cells were then washed twice, followed by sorting of the epithelial cells (EpCAM+ / CD31- / CD45-) by fluorescence-activated cell sorting at the Cytometry and Cell Sorting Core at BCM.
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| Contributor(s) |
Kim B, Zhang S, Huang Y, Park J |
| Citation(s) |
38796854 |
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| Submission date |
Apr 18, 2023 |
| Last update date |
Aug 01, 2024 |
| Contact name |
Bongjun Kim |
| E-mail(s) |
arrice86@gmail.com
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| Organization name |
MD Anderson Cancer Center
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| Department |
Experimental Radiation Oncology
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| Lab |
Jae-Il Park Lab
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| Street address |
6565 MD Anderson BLVD
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| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA956923 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE229982_RAW.tar |
460.3 Mb |
(http)(custom) |
TAR (of H5AD) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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