DNA methylome analysis of leader cells and follower cells derived from the H1299 NSCLC cell line

Tumor heterogeneity drives disease progression, treatment resistance, and patient relapse, yet remains largely under-explored in invasion and metastasis. Here, we investigated heterogeneity within collective cancer invasion by integrating DNA methylation and gene expression analysis in rare purified lung cancer leader and follower cells. Our results showed global DNA methylation rewiring in leader cells and revealed the filopodial motor MYO10 as a critical gene at the intersection of epigenetic heterogeneity and 3D collective invasion. We further identified JAG1 signaling as a novel upstream activator of MYO10 expression in leader cells. Using live cell imaging, we discovered that MYO10 drives filopodial persistence necessary for micropatterning extracellular fibronectin into linear tracks at the edge of 3D collective invasion exclusively in leaders. Our data fit a model where epigenetic heterogeneity and JAG1/Notch signaling jointly drive collective cancer invasion through MYO10 upregulation in epigenetically permissive leader cells, which induces filopodia dynamics necessary for linearized fibronectin micropatterning. Using the H1299 NSCLC cell line, parental cells, follower cells, and leader cells were isolated from collectively invading spheroids in Matrigel as previously described (Konen et al., Nat Comm, 2017; doi: 10.1038/ncomms15078). DNA methylation status was assessed in triplicate samples of H1299 parental, leader and follower cells. Bisulphite converted DNA from the 9 samples were hybridized to the Illumina Infinium MethylationEPIC array.