Exploring the influence of fibrotic tumour microenvironment on lung cancer development using in vitro 2D y 3D cell culture models

Lung cancer (LC) is the leading cause of cancer death. The risk of LC is increased up to 5-fold in patients with pulmonary fibrosis, and these patients have a worse prognosis as diagnosis and treatment become more complicated. We hypothesize that the fibrotic tumour microenvironment (TME) modulates tumoral cell growth and triggers transcriptomic changes. In this project, we cultured in 2D and 3D lung tumoral cell lines (adenocarcinoma NSCLC cell lines: H23 and H441 and squamous NSCLC: EBC1 cell line) under profibrotic conditions (presence of TGF-β, PDGF, FGF2, IL-4, and IL-13). In the 3D cultures, we performed multicellular spheroids combining lung tumoral cells with primary lung fibroblasts and macrophages. Cell proliferation was measured by flow cytometry and expression of genes involved in regulating the immune pathways by qPCR. our data show a decrease in the proliferation rate after 72 h of growing them in fibrotic conditions in 2D conditions. The 3D multicellular co-culture experiments under profibrotic conditions alters tumoral cell behaviour and growth. Adenocarcinoma tumoral cells cultured in profibrotic conditions exhibited an altered immune marker expression, with a statistically significant decreased expression in PDL-1 and STING (stimulator of interferon genes) in fibrotic conditions, suggesting that a fibrotic TME may alter the response of tumoral cells to immunecheckpoint therapies. Unexpectedly, our data highlight the significant impact of a fibrotic microenvironment on tumoral cell growth and its immune marker expression. A deeper study of the signalling mechanisms and the transcriptomic changes in tumoral cells is needed to set up new therapies for these patients.