In Vitro 3‐Dimensional Modeling of Tumor Microenvironment Reveals Host/Tumor Influence on Progression in Non‐Small Cell Lung Carcinoma

Abstract

BackgroundLung cancer is the leading cause of cancer related death in the United States and worldwide. Five‐year survival rates for non‐small cell lung carcinoma patients have not improved in decades, and a vast majority of patients succumb to metastasis. Identification of therapeutic targets for advanced stage disease is essential for the development of novel therapies to halt the progression of this devastating disease. However, current 2‐dimensional (2D) in vitro models are not reflective of the tumor microenvironment and are inadequate in identifying these targets. We previously reported that lung carcinoma cells grown on a human lung fibroblast derived 3‐dimensional (3D) extracellular matrix (ECM) display differences in cell morphology, proliferation, migration, and survival as compared to when they are grown in 2D. Our 3D in vitro model provides the capability to study the interaction of lung carcinoma cells with the tumor microenvironment and characterize host microenvironment effects on cancer progression.ObjectiveDetermine how ECM interactions with lung adenocarcinoma cells can promote hallmarks of cancer metastasis in an in vitro model.MethodsHuman WI‐38 fibroblasts and mouse primary fibroblasts were maintained for 8 days and then decellularized to produce ECM. Human lung adenocarcinoma cell lines A549 and mouse KrasLSL‐G12D/+;p53flox/flox lung adenocarcinoma cell lines were then grown on these matrices (3D) or plastic (2D). ECM to cell interactions were disrupted via inhibitors of ‘outside‐in’ signaling including integrins, focal adhesion kinase, src family kinases, and cytoskeletal remodeling. Cancer cell growth was assessed by AlamarBlue and markers of cancer progression were assessed by western blot analysis. Migration and invasion were assessed by matrigel scratch wound assay.ResultsCarcinoma cells grown on 3D matrices are significantly more invasive as compared to 2D. The increased invasion of A549 cells grown in 3D ECM was attenuated with inhibitors of Src family kinases and focal adhesion kinases. The altered phenotype observed when A549 cells are grown on 3D ECM was not specific to the source of the matrix as it was also observed when ECM was generated by primary fibroblasts derived from mice. Growth of cancer cells on mouse derived ECM with cancer conditioned media altered expression of known markers of EMT.ConclusionsUsing our 3D ECM model, we demonstrate that we are able to study ECM‐to‐cell interactions in vitro in a way that is not possible for traditional 2D models. These interactions play a pivotal role in determining the fate of cancer cells. Additionally, our model is expandable to primary fibroblasts derived from mice, which allows manipulation of the host microenvironment to study mechanisms of cancer progression in a physiologically relevant model. An understanding of how remodeling of the tumor microenvironment is involved in late stage disease will allow development of preventative and curative therapeutic strategies to halt tumor progression and metastasis.Support or Funding InformationThis research was supported by funds from National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01‐DK093462, to L.J.S], National Cancer Institute [Grant R01‐CA193220, to L.J.B], and startup funds from James Graham Brown Cancer Center, University of Louisville School of Medicine (to L.J.B. and L.J.S.).