Prognostic significance of an invasive leader cell–derived mutation cluster on chromosome 16q

Abstract

Background Intratumoral heterogeneity is defined by subpopulations with varying genotypes and phenotypes. Specialized, highly invasive leader cells and less invasive follower cells are phenotypically distinct subpopulations that cooperate during collective cancer invasion. Because leader cells are a rare subpopulation that would be missed by bulk sequencing, a novel image‐guided genomics platform was used to precisely select this subpopulation. This study identified a novel leader cell mutation signature and tested its ability to predict prognosis in non–small cell lung cancer (NSCLC) patient cohorts. Methods Spatiotemporal genomic and cellular analysis was used to isolate and perform RNA sequencing on leader and follower populations from the H1299 NSCLC cell line, and it revealed a leader‐specific mutation cluster on chromosome 16q. Genomic data from patients with lung squamous cell carcinoma (LUSC; n = 475) and lung adenocarcinoma (LUAD; n = 501) from The Cancer Genome Atlas were stratified by 16q mutation cluster (16qMC) status (16qMC+ vs 16qMC−) and compared for overall survival (OS), progression‐free survival (PFS), and gene set enrichment analysis (GSEA). Results Poorer OS, poorer PFS, or both were found across all stages and among early‐stage patients with 16qMC+ tumors within the LUSC and LUAD cohorts. GSEA revealed 16qMC+ tumors to be enriched for the expression of metastasis‐ and survival‐associated gene sets. Conclusions This represents the first leader cell mutation signature identified in patients and has the potential to better stratify high‐risk NSCLC and ultimately improve patient outcomes.

A live-cell platform to isolate phenotypically defined subpopulations for spatial multi-omic profiling

“…6B). Previously established leader-and follower-specific gene mutations were used to complement positional phenotypes to label each single cell, enabling for the first time, the combination of phenotypic, transcriptomic, and mutational analysis of single cells during active collective invasion 29,30 . We selected mutation loci from the list of leader-and follower-specific genes and verified that they had sufficient coverage in the single-cell sequences.…”

“…These results indicate that our NSCLC spatial localization is a coordinated patterning driven by genomic and epigenetic cellular profiles. At the RNA and protein levels, we found that underlying differences in their filopodia dynamics driven by a Jag1-Myo10 signaling axis further contribute to the stark differences in invasive capacities of the leaders and followers [28][29][30] . Similarly, invasive chains harbor metabolic heterogeneity, in which trailing followers are highly glycolytic and leaders depend upon mitochondrial respiration 27 .…”

“…Combining SaGA and multiple non-small cell lung cancer (NSCLC) lines, cell(s) were photoconverted based upon their spatial positioning within the collectively invading pack to isolate leader (front of the pack) and follower (trailing behind leaders) subpopulations 26 . To assess phenotypic stability overtime, cells were sorted for propagation and long-term phenotypic and transcriptomic analysis [26][27][28][29][30] . Epigenetic differences were assessed by performing a DNA methylation array, where cells were sorted for immediate DNA extraction and epigenetic analysis 26,28 .…”

“…Using the H1299 NSCLC line, we found that leaders and followers isolated during 3D collective invasion have distinct genotypic, epigenetic, and phenotypic differences, and are phenotypically stable over many passages [26][27][28][29][30] . By mapping our bulk RNA sequencing results to the human reference genome Hg19 (GRCh37) and various filtering steps, we identified 14 distinct missense mutations between leaders and followers 30 .…”

“…Utilizing SaGA-derived leader and follower cells, we performed multi-omic analysis to extrapolate DNA methylation status and bulk transcriptomic profiling 26, [28][29][30] . We established that leaders and followers maintain stable differences at the epigenetic, genetic, metabolomic, and transcriptomic level [26][27][28][29][30] . Similarly, propagation of isolated leader and follower subpopulations generate stable phenotypes for over 30 passages (Fig.…”