Project Summary Collective invasion is a major mode of metastasis observed in patients across most solid tumor types. How the collective invasion pack operates, communicates, and navigates as a single cohesive unit remains unclear. To address this, we published on an image-guided genomics platform to isolate any living cell(s) within a collective invasion pack, and expand the population for genomic and molecular analysis, a technique termed Spatiotemporal Cellular & Genomic Analysis (SaGA). We used SaGA to dissect the molecular, epigenetic, and genomic profiles of leader and follower cells invading as a hierarchical cohesive unit. To determine how epigenetic reprogramming drives this phenotypic heterogeneity, we deconstructed the collective invasion pack using SaGA, then integrated genome-wide promoter methylation and transcriptome data to define differentially methylated regions within the leader and follower phenotypes. We observe global epigenomic re-wiring in leader cells supporting an epigenetic basis for the phenotypic heterogeneity within the collective invasion pack. We then identified Myo10 (myosinX) as a top differentially methylated and expressed gene, where the leader cell promoter is hypomethylated, and leaders in several lung cancer lines overexpress Myo10. Myo10 is a canonical modulator of filopodia elongation and we show it drives filopodia elongation, collective invasion, leader cell-driven fibronectin micropatterning (fibrillogenesis), and is transcriptionally activated by Jag1/Notch. We will use this information to test a mechanistic model with the overarching hypothesis that Myo10 activation via promoter hypomethylation in leader cells drives filopodia-based micropatterning of fibronectin to create a leader cell-driven collective invasion path. We propose that this leads to an invasive advantage for lung cancer cells resulting in metastatic disease. In Aim 1 we test the model that Myo10 hypomethylation in leaders allows for Jag1/Notch1- driven transcriptional activation, driving filopodia elongation, and fibronectin micropatterning. In Aim 2 we test how this collective invasion pathway impacts metastasis using in vivo metastasis models and the first patient- derived leader cells. Throughout, we leverage unique resources developed here including SaGA-derived cell lines, ex vivo imaging, and patient-derived lung cancer leader cells. We speculate that these data will provide mechanistic insight into collective invasion and translational value towards understanding lung cancer patient leader cell biology.

Narrative Collective invasion is a major mode of metastasis observed in patients across most solid tumor types. We developed a new imaging-based technique to obtain molecular and epigenetic data of specialized cell types within the lung cancer collective invasion pack. We will use this information to probe the mechanistic underpinnings of Myo10 function within the collective pack.