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.

Exosomes, which are one of the smallest extracellular vesicles released from cells, have been shown to carry different nucleic acids, including microRNAs (miRNAs). miRNAs significantly regulate cell growth and metabolism by posttranscriptional inhibition of gene expression. The rapidly changing understanding of exosomes’ formation and function in delivering miRNAs from cell to cell has prompted us to review current knowledge in exosomal miRNA secretion mechanisms as well as possible therapeutic applications for personalized medicine.

MicroRNA (miRNA) are small non-coding RNA molecules that posttranscriptionally effect mRNA stability and translation by targeting the 3′-untranslated region (3′-UTR) of various transcripts. Thus, dysregulation of miRNA affects a wide range of cellular processes such as cell proliferation and differentiation involved in organ remodeling processes. Divergent miRNA patterns were observed during chronic liver diseases of various etiologies. Chronic liver diseases result in uncontrolled scar formation ending up in liver fibrosis or even cirrhosis. Since it has been shown that miR-29 dysregulation is involved in synthesis of extracellular matrix proteins, miR-29 is of special interest. The importance of miR-29 in hepatic collagen homeostasis is underlined by in vivo data showing that experimental severe fibrosis is associated with a prominent miR-29 decrease. The loss of miR-29 is due to the response of hepatic stellate cells to exposure to the profibrogenic mediators TGF-β and PDGF-BB. Several putative binding sites for the Smad proteins and the Ap1 complex are located in the miR-29 promoter, which are suggested to mediate miR-29 decrease in fibrosis. Other miRNA are highly increased after profibrogenic stimulation, such as miR-21. miR-21 is transcriptionally upregulated in response to Smad-3 rather than Smad-2 activation after TGF-β stimulation. In addition, TGF-β promotes miR-21 expression by formation of a microprocessor complex containing Smad proteins. Elevated miR-21 may then act as a profibrogenic miRNA by its repression of the TGF-β inhibitory Smad-7 protein.

In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC. HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3′-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. The 3′-UTR of the collagen-1 and −4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively.

Background: MicroRNAs have recently taken centre stage as short non-coding RNAs that regulate mRNA expression. Aim/Methods: To assess the feasibility of using microRNA techniques on routinely processed tissues, the accessibility of two representative microRNAs was examined by real-time quantitative PCR in 86 human formalin-fixed paraffin-embedded (FFPE) samples from liver, breast, bone marrow, lymphatic tissues and colon. Murine liver was used to analyse the influence of fixation time and different fixatives. Results: High-quality microRNA was successfully extracted from routinely processed formalin-fixed tissues, resembling PCR amplification results from snap-frozen material analysed in parallel. While fixation time did not affect microRNA accessibility, non-buffered formalin or fixative supplements such as glutaraldehyde influenced PCR results. Storage of human tissues for up to 7 years did not cause a significant deterioration of microRNA. However, microRNA quality in human archival material following routine processing 10–20 years ago was decreased. Oxidation by ambient air during storage and fixation in non-buffered formalin is a possible reason for loss of microRNA quality. Conclusion: The assessment of microRNAs in readily obtained formalin-fixed paraffin-embedded samples is a highly promising tool in molecular pathology when similarly treated samples are analysed. Therefore, microRNA analyses will gain wider acceptance as an adjunct to morphological tissue assessment in routine pathology and retrospective studies.