Ammar, Amany; (2024) Using alveolar organoids to chart tobacco-induced genetic damage and impact on cell fate. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In lung cancer, the interplay between mutation, clonal expansion, and selective pressures is linked to tobacco smoking. While ex-smokers appear to have falling rates of lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD) rates are relatively preserved. In my thesis work, I have worked with mathematicians and geneticists to confirm this epidemiological observation and then try to demonstrate that rather than putative changes in smoking habits on cigarette make-ups, this is in fact due to the regenerative capacity of the lungs. We first confirmed the epidemiological difference in rates of histological types over time, analysing data from over 650,000 individuals. I then generated a novel organoid system for alveolar type II (AT2) cells, stem cells of the lung alveoli and a potential cell of origin of LUAD, allowing me to generate enough genomic DNA from a single cell to then assess over 800 whole genomes of AT2 cells. This revealed high mutation burdens, regardless of smoking cessation. This is in contrast to our laboratory’s previous work showing LUSC progenitor basal cells demonstrate selection of cells with near-normal mutational burden after quitting smoking. I confirmed these findings, working with the Sanger Institute, by profiling AT2 cells using targeted nanorate sequencing (tNanoSeq) in 37 individuals. We demonstrate a clonal landscape with a limited number of cancer driver genes under positive selection. Integrating genomic and epidemiological data in a classical multistage model of carcinogenesis, we find that differences in LUAD and LUSC incidence in ex-smokers are rooted in the absence of protected unmutated AT2 stem cells in the alveoli. Having developed stable single-cell derived organoids, I developed these as a tool to study human cancer. I genetically engineered normal human alveolar organoids by lentiviral transduction. The findings demonstrate that normal human alveolar organoids are amenable to genetic modifications and could express the transgene (CopGFP) for a considerable duration. I then incorporated the oncogenic KRASG12D mutation into the lentiviral vector and transduced the organoids with the modified construct. The engineered organoids expressed the KRASG12D oncogene and exhibited a larger size compared to control organoids, suggesting that the KRASG12D mutation may increase the proliferation rate of human AT2 cells and also demonstrating the potential of alveolar organoids to mimic oncogenic traits in vitro, paving the way for emerging next-generation tools to study LUAD progression in human cells.

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