Effect of Myosin 10 or Jagged 1 knockout on gene expression in H1299 Non-small cell lung cancer leader cells.

While minimizing lung cancer metastasis can improve overall survival, few models exist where subpopulations with stable high and low metastatic potential can be easily and reliably isolated. Our previous work characterized two stable subpopulations, termed Leaders and Followers, from the H1299 Non-Small Cell Lung Cancer model. H1299 Leaders and Followers have different transcriptional profiles, biomarkers, genomic methylation patterns, metabolic states, and morphologies.Of note, H1299 Leaders express high levels of Jagged1 (JAG1) and Myosin 10 (MYO10) and drive collective invasion and metastasis. To determine how JAG1 and MYO10 contribute to Leader cell biology and lung cancer metastasis, we utilized CRISPR-Cas9 to generate edited pools of H1299 cells targeting either JAG1 or MYO10. Fluorescence-activated cell sorting using a Leader-Follower biomarker, IL13R2, was used to generate single cell derived candidate clones. Candidate clones were screened using western blot for Leader and Follower cell biomarkers (JAG1, MYO10, HTATIP2 and IL13R2) followed by Sanger sequencing of candidate clones was also conducted to confirm target gene status. Using this method, 2 JAG1 knockout, 2 MYO10 knockout, 1 MYO10 knockdown and, 2 wildtype MYO10 JAG1 H1299 single-cell derived clones were generated.