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Abstract

Multicellular organisms rely on the concerted interaction of a multitude of cells, which are often highly specialised and give rise to complex tissues. As vastly different cellular phenotypes emerge from the same genotype that is shared across all cells of an organism, the transcriptome represents a key mediator driving different cell types and cell states that give rise to functional tissues. These are also subject to environmental factors or intrinsic changes that may disrupt homeostasis and lead to disease. In the human lung, the effects of tobacco smoke exposure, still the greatest risk factor for lung cancer, have not been fully resolved at the cellular level. Moreover, cellular heterogeneity may be significant for the emergence of lung cancer in never smokers, a growing proportion of global cases. A focused investigation of cellular heterogeneity in the healthy lung and lung cancers is therefore highly warranted. During the last decade, technological advancements have made it possible to interrogate the transcriptome of single cells by novel next generation sequencing approaches. While previous studies were limited to averaging transcriptome information over many cells, single cell RNA sequencing (scRNA-seq) technologies are now enabling the investigation of cellular phenotypes in healthy and diseased tissues at unprecedented resolution. In this thesis, I adapt different scRNA-seq technologies to process fresh or biobanked samples from different tissues and species, thus enabling comparisons across diverse origins. We identify specific advantages, limitations and experimental challenges associated with each technology. I then perform a comprehensive single-cell transcriptomics study of healthy lung and lung adenocarcinoma (LADC). Based on twelve healthy lung samples, we generate a reference cell atlas that provides a rich resource for investigating cellular diversity in the human alveolar lung. Its utility is demonstrated by probing the expression of genes that are implicated in host cell entry of SARS-CoV-2 virus, thereby vi contributing to our understanding of coronavirus infections. By comparing single cell profiles from smokers and never smokers, we also resolve the involvement of distinct cell types in the maintenance of an inflammatory state in smoker lungs, and we identify key mediators of inflammatory processes induced by tobacco smoke exposure in fibroblasts and endothelial cells. To investigate cell type diversity and microenvironment interactions in LADC, I analyse 26 tumour tissue samples and resolve functional malignant cell subpopulations linked by a differentiation hierarchy in both smokers and never smokers. They comprise proliferating and intermediate undifferentiated cells as well as two differentiated tumour cell states implicated in cancer progression and invasiveness. Distinct macrophage and fibroblast subpopulations which contribute to a tumourigenic environment are also detected. A subset of proliferating tumour cells show differential immune modulating activity dependent on smoking status, with implications for future treatment approaches. Taken together, these results provide a comparison of rapidly developing scRNA-seq technologies for use in further studies and demonstrate their utility to dissect cellular heterogeneity and identify transcriptional programmes in the healthy and diseased lung. By applying these technologies, I add to our understanding of SARS-CoV-2 entry into human lung cells, define the alveolar lung cell types affected by tobacco smoke exposure, and provide deeper insight into cellular heterogeneity of LADC and the tumour microenvironment. These findings represent a valuable reference for future translational studies.