Exploring the impact of the fibrotic microenvironment on lung tumors using a 3D multicellular spheroid model

Lung cancer (LC) is one of the most common cancers and has the highest mortality rate. The risk of developing LC increases more than 7-fold in patients with chronic respiratory diseases, such as pulmonary fibrosis, leading to a worse prognosis and making treatment more complicated. Our hypothesis is that the fibrotic tumor microenvironment and cell-cell communication modulate tumor cell growth and modify their transcriptome We recreated tumor growth <italic>in vitro</italic> by culturing spheroids with non-small cell lung cancer cell lines (H23-adenocarcinoma, EBC1-squamous) alone or in multicellular spheroids co-cultured with human lung fibroblasts and macrophages under fibrotic (TGF-β, PDGF, FGF2, IL-4 and IL-13) and non-fibrotic conditions. We measured cell proliferation by flow cytometry, gene expression by bulk-RNA-sequencing and qPCR, and measured cell-cell interactions using microscopy. We observed that tumoral cells from mono- and multicellular spheroids under fibrotic conditions showed decreased proliferation in both LC lines. Fibroblasts in EBC1 co-culture and macrophages in H23 coculture reversed these effects. Transcriptomic data analysis revealed alterations due to fibrotic conditions in genes related to cell cycle and immune response such as CD73 that was significantly increased in both LC lines or PD-L1 that was upregulated in monocellular spheroids but decreased in the multicellular. Our data highlight the impact of the fibrotic microenvironment on tumoral cell functionality and its response to potential immunotherapies. For this reason, it is necessary to further study the altered pathways in tumor cells to identify new therapeutic options for this type of patients.