Lung cancer is a highly heterogeneous disease. Cancer cells and cells within the tumor microenvironment together determine disease progression, as well as response to or escape from treatment. To map the cell type-specific transcriptome landscape of cancer cells and their tumor microenvironment in advanced non-small cell lung cancer (NSCLC), we analyze 42 tissue biopsy samples from stage III/IV NSCLC patients by single cell RNA sequencing and present the large scale, single cell resolution profiles of advanced NSCLCs. In addition to cell types described in previous single cell studies of early stage lung cancer, we are able to identify rare cell types in tumors such as follicular dendritic cells and T helper 17 cells. Tumors from different patients display large heterogeneity in cellular composition, chromosomal structure, developmental trajectory, intercellular signaling network and phenotype dominance. Our study also reveals a correlation of tumor heterogeneity with tumor associated neutrophils, which might help to shed light on their function in NSCLC.

Vertebrate muscle differentiation is coordinated by an intricate network of transcription factors requiring proliferating myogenic precursors to withdraw irreversibly from the cell cycle. Recent studies have implicated a large number of microRNAs exerting another layer of control in many aspects of muscle differentiation. By annealing to short recognition sequences in the 3-untranslated region, microRNAs attenuate target gene expression through translation repression or mRNA degradation. Here, we show that miR-214 promotes myogenic differentiation in mouse C2C12 myoblasts at a step preceding the induction of p21 and myogenin. Blocking miR-214 function with a 2-O-methylated double-stranded inhibitor maintained C2C12 cells in the active cell cycle, thereby inhibiting the myogenic differentiation. By global gene expression profiling, we identified the proto-oncogene N-ras as one of miR-214 targets. Furthermore, manipulating the N-Ras level with small interfering RNA or adenovirus-mediated forced expression either augmented or attenuated the effect of miR-214, respectively. Thus, our data uncovered a novel microRNA-mediated mechanism that controls myogenic differentiation.The vertebrate skeletal muscle is developed from mesodermal stem cells that are committed to a muscle fate within paraxial somites, giving rise to immature myoblasts (1-4). Committed myoblasts are proliferating progenitors (5) that must exit from the cell cycle before myogenic regulatory factors such as MyoD and Myf5 activate myocyte enhancer factors and other downstream muscle-specific genes to drive the formation of multinucleated myotubes and eventually the contractile muscle fibers (6 -9). This process can be recapitulated in vitro with cell culture systems, the most widely used of which is the mouse C2C12 cell (10, 11). In proliferating myoblasts, the MyoD targets of myogenic genes are repressed through chromosome remodeling and epigenetic histone modifications (12, 13). Under the influence of extracellular signals in developing muscle tissues or upon serum depletion in culture systems, MyoD orchestrates an orderly exit from the cell cycle, involving concerted interplays between myogenic regulatory factors and the cell cycle machinery (7,9,13,14). The retinoblastoma gene product Rb and its homologue p107 (15-17) are essential in initiating an irreversible withdrawal from the cell cycle by upregulating cyclin-dependent kinase inhibitor p21 (18,19). Certain oncogene products including H-and N-Ras exhibit potent inhibition on myogenic differentiation by blocking Rb function (20).In addition to the complex network of protein-encoding genes, recent molecular and genetic studies have uncovered a myriad of microRNAs (miRNAs) 2 that exert a different layer of control over myogenic differentiation (21-23). miRNAs are a class of small noncoding RNAs that are synthesized as primiRNAs from either dedicated transcription units or introns of protein-encoding genes (24 -26). Pri-miRNAs are processed in the nucleus by the RNase Drosha, yielding precursor mi...

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