Abstract 4590: Dissecting the biology of leader and follower cells in collective cancer invasion

Abstract

Abstract Previous work has demonstrated heterogeneity within collectively invading packs of lung cancer cells, including leader and follower cells that cooperate to facilitate invasion into the microenvironment. In order to better characterize the genetic differences between these two distinct cell types, we performed RNA-seq on purified populations of leader and follower cells from the H1299 non-small cell lung cancer cell line. Through this analysis, we identified that leaders and followers each contain distinct mutational and gene expression profiles, despite originating from the same parental cell line. Importantly, we identified 17 point mutations found uniquely in leaders and 18 point mutations found uniquely in followers, thus representing the first known compilation of leader- and follower-enriched genetic variants. Notable leader-enriched mutated genes included NAE1, NUP93 and ACTR3, while notable follower-enriched mutated genes included NADK, NDUFS1 and LEO1, suggesting possible roles for these genes in the biological function of leader and follower cells. After validating these mutations in the genomic DNA of leaders and followers, we sought to determine whether these mutational signatures are correlated with leader and follower gene expression markers within individual cells in a heterogeneous tumor cell population. To this end, we performed single-cell RNA-seq on H1299 parental, leader and follower cells isolated directly from collectively invading packs in a 3-D matrix. Hierarchical clustering and tSNE analysis based upon most variably expressed genes revealed four distinct cell clusters; two expressing higher levels of leader cell genes including MYO10 and JAG1 (clusters 1 and 4), and two expressing higher levels of follower genes including IL13RA2 and HTATIP2 (clusters 2 and 3). Interestingly, clusters 1 and 4 are composed exclusively of cells with leader mutational profiles, while nearly all cells in clusters 2 and 3 contain follower mutational profiles, suggesting that these mutations could be driving the differential gene expression, and ultimately the unique biological behaviors, of leaders and followers. In addition, clusters 1 and 2 display higher levels of proliferative markers compared with clusters 3 and 4, an indication that there are proliferative and non-proliferative subpopulations within the leader and follower populations. Taken together, these data give us new insight into the multiple levels of heterogeneity that exist within an invasive tumor cell population, suggest novel drivers of leader and follower cell biology in collective invasion, and open the door to new potential strategies for targeting and inhibiting metastasis in human lung cancer. Citation Format: Brian A. Pedro, Jessica Konen, Emily Summerbell, Janna K. Mouw, Manali Rupji, Bhakti Dwivedi, Jeanne Kowalski, Paula M. Vertino, Adam I. Marcus. Dissecting the biology of leader and follower cells in collective cancer invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4590.