Influenza-Induced Remodelling Accelerates the Development of Non-Small Cell Lung Cancer
Creators: name: Reynolds, David
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 320103
subject: Respiratory diseases

description: 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.