Influenza-Induced Remodelling Accelerates the Development of Non-Small Cell Lung Cancer

Lung cancer, particularly non-small cell lung cancer (NSCLC), remains a leading cause of global mortality. While smoking is an undeniable risk factor, the underlying mechanism of NSCLC development remains unknown, leaving us with limited therapeutic options. The prevailing model of tumourigenesis assumes a stepwise accumulation of genetic alterations, each of which confers a selective growth advantage by activating a specific signalling pathway that drives cancer progression. However, the abundance of genetic alterations observed in both healthy individuals and non-tumour cells in cancer patients suggests a more nuanced picture, where additional factors like the tumour microenvironment (TME) play a role. We propose that injury-induced remodelling promotes changes in the lung microenvironment that promotes the expansion of pre-existing mutated clones. Notably, influenza infection, which affects roughly one billion people annually and is known to cause lung remodelling, has shown potential links to increased lung cancer risk. However, the precise impact of influenza on cancer progression remains largely unknown. The TME is a complex network of cells surrounding a tumour, including stromal cell types that can significantly influence tumour cell growth. Despite this, our understanding of the specific cell types within the TME and their role in NSCLC development remains limited. This lack of detail extends to the term "cancer-associated fibroblasts" (CAFs), which encompasses a diverse population of stromal cells within the TME with functions that can either promote or suppress tumour growth. By investigating how acute influenza A virus (IAV) infection alters the composition of the TME, we aim to gain insights into its potential influence on NSCLC progression, particularly the possibility of an injury-induced tumour promotion stage in lung tumour formation. To achieve this, we utilised mice with genetically engineered floxed alleles of the Lkb1 and Pten genes, which are frequently mutated in NSCLC. This model allows us to investigate whether influenza infection can accelerate NSCLC development in cells with pre existing mutations and explore the interplay between influenza-induced lung injury, TME development, and tumour progression. The first study used a mild strain of influenza to induce aberrant epithelial remodelling in a mouse model of acute lung injury. Our goal was to identify the role of stromal cell subtypes in driving this phenomenon. We discovered that a mild strain of influenza led to aberrant epithelial remodelling, which was accompanied by significant alterations in the stromal compartment of the lung, persisting for at least 21 days post infection (dpi). As previously described, the epithelial remodelling was characterised by the bronchiolisation of the alveolar epithelium with dysplastic expansion of basal cells and differentiation of ciliated cells and goblet cells in the lung parenchyma. This was also associated with extensive collagen 1 deposition and fibrotic remodelling indicative of maladaptive stromal cell activation. We developed a flow cytometry strategy to subset stromal cell populations from the mouse lung and identified unique epithelial supportive stromal subsets, as demonstrated by their ability to support epithelial growth in vitro using a 3D lung organoid culture system. These include a FAPα+ resident mesenchymal stem cell population (rMSCs) and a highly supportive IL11+ population we termed auxiliary fibroblasts. In situ, we observed FAPα+ and IL11+ cells localised to areas of aberrant epithelial remodelling and fibrosis 21 dpi following influenza infection. Of note, only animals that had prolonged epithelial remodelling displayed fibrosis with co-localisation of high numbers of FAPα+ and IL11+ cells. This study suggests that stromal cell subsets may be key players in promoting stem cell-mediated regeneration and remodelling after influenza-induce lung injury. Interestingly, the stromal cell subsets that we identified in this study share striking similarities with CAF subtypes that are involved in chronic lung diseases and NSCLC. We next looked at the impact of influenza infection on tumour promotion using our transgenic mouse model of NSCLC, which involved deletion of floxed Lkb1 and Pten genes in the lung by intranasal delivery of adenovirus expressing Cre recombinase. This study showed that influenza infection can accelerate NSCLC development from lung epithelial cells with pre existing mutations in Pten and Lkb1 (genes known to be associated with human NSCLC) as well as tumour progression (increased size). Non-small cell lung cancer (NSCLC) can be sub divided into squamous cell carcinoma (SCC) and adenocarcinoma (ADC). In this model we observed heterogeneity in tumour phenotypes, with smaller tumours being predominately a mucinous ADC phenotype and larger tumours being a mixed or SCC phenotype. Additionally, in our long-term study control group, we discovered that mild influenza infection can lead to chronic aberrant epithelial remodelling persisting up to 40 weeks post infection (wpi), a previously unknown finding that supports the concept that early life events can have significant impact on lung health later in life. The final study examined the relationship between influenza infection, FAPα+ and IL11+ stromal cells and the establishment of a tumour promoting TME. Immunostaining of lung tissue sections from the Lkb1/Pten-deficient NSCLC mouse model revealed that FAPα+ and IL11+ stromal cells localised to the leading and developing edges of tumours at all stages of development. Notably, their location differed from pro-fibrotic myofibroblast populations marked by α-smooth muscle actin (ASMA). These results suggest that FAPα+ and IL11+ supportive fibroblasts (rather than ASMA+ cells) are key players in establishing a tumour promoting TME. This research has identified novel stromal cell populations in the lung that, in response to influenza induced injury, play a critical role in establishing both a regenerative microenvironment and a tumour promoting TME. These findings represent a significant breakthrough in understanding the link between influenza infection and lung cancer development. By focusing on the TME and specific stromal cell populations involved in epithelial regeneration, we have shed light on the underlying mechanisms at play in lung tissue regeneration, remodelling and tumour formation. Furthermore, this work underscores the potential long-term consequences of lung infections, including promotion of lung cancer.