Gene expression profiles at single cell level of lung epithelial cells from the Cracd WT and Cracd KO mice. Gene expression profiles at single cell level of lung epithelial cells from the Cracd WT and Cracd KO mice

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. 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.