Abstract 309: Longitudinal monitoring of cell proliferation and cytotoxicity in a biomimetic 3D culture model for lung cancer using native extracellular matrix scaffolds
Authors: Tapias Luis F. , Lanuti Michael , Ren Xi , Gilpin Sarah E. , Wei Lan , Fuchs Bryan C. , Tanabe Kenneth K. , Ott Harald C. 
Published: August 2015

Abstract
Introduction: Tissue-engineered 3D cell culture techniques have gained popularity in cancer research as they recapitulate tissue architecture, facilitate stroma formation, and promote complex cell-cell and cell-extracellular matrix (ECM) interactions. Decellularized whole-organ scaffolds may represent the ultimate ECM analogue for this purpose. However, methods to assess cell behavior or viability within these tumor constructs are lacking.
Methods: Lungs from Sprague Dawley rats were subjected to perfusion-decellularization via the pulmonary artery with 0.1% sodium dodecyl sulfate and 1% Triton X-100 to obtain decellularized whole-lung ECM scaffolds. Decellularized lung scaffolds were placed in a bioreactor to allow perfusion of cell-specific media through the pulmonary artery. The human lung cancer cell lines SW1573 (KRAS mutant) and PC9 (EGFR mutant) were seeded into the scaffolds by means of tracheal delivery and were cultured for a total of 7 days. A resazurin reduction perfusion assay was performed on days 1, 3, 5 and 7 of culture to evaluate cell viability by diluting a resazurin-based reagent with culture medium and perfusing it via the pulmonary artery. The net fluorescence increase at the end of the assay was used to estimate the number of viable cells. Some tumor-ECM constructs were treated with erlotinib starting on day 3 of culture. At 7 days, tissue was collected and analyzed by histology and immunostaining for Ki67 and activated caspase 3.
Results: The human lung cancer cell lines PC9 and SW1573 showed engraftment and macroscopic tumor nodule formation when cultured on native lung ECM scaffolds under biomimetic 3D conditions. The resazurin reduction perfusion assay permitted the longitudinal and non-destructive estimation of cell viability within these 3D tumor constructs, therefore allowing the generation of proliferation curves using this model. Doubling times for PC9 and SW1573 cells under 3D biomimetic conditions were approximately 4.2 days and 4.7 days. Additionally, this method was able to detect sensitivity and relative resistance to erlotinib. After introduction of erlotinib on day 3, this assay reliably identified decreasing viable cell numbers in PC9-seeded scaffolds (∼23% vs. before treatment) consistent with the genotypic background of this cell line (EGFR exon 19 deletion). On the other hand, this method detected ongoing proliferation of SW1573 cells after erlotinib (∼148% vs. before treatment), consistent with known relative resistance of this cell line to this agent, which correlated with caspase 3 expression.
Conclusions: Decellularized native lung ECM scaffolds can serve as a more realistic 3D model for the study of processes related to lung cancer progression or for therapeutic experimentation. The methods presented here will prove useful to study cancer proliferation and cell viability under these 3D biomimetic culture conditions.
Citation Format: Luis F. Tapias, Michael Lanuti, Xi Ren, Sarah E. Gilpin, Lan Wei, Bryan C. Fuchs, Kenneth K. Tanabe, Harald C. Ott. Longitudinal monitoring of cell proliferation and cytotoxicity in a biomimetic 3D culture model for lung cancer using native extracellular matrix scaffolds. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 309. doi:10.1158/1538-7445.AM2015-309