Abstract 326: Differences in RNA expression and chemosensitivity in 2D versus 3D non-small-cell lung cancer cultures
Authors: Gamerith Gabriele , Rainer Johannes , Amann Arno , Koeck Stefan , Lorenz Edith , Zwierzina Heinz , Huber Julia M. 
Published: August 2015

Abstract
Introduction: Increasing efforts to integrate tumour-stroma interaction and 3-dimensional structures in innovative in-vitro models were made within the last years. We established a 3D co-culture system in non-small-cell lung cancer applicable for drug testing. In this work, we present data on the differences in RNA expression and drug sensitivity in the 2D and 3D culture system.
Methods: A549 and Colo699 NSCLC cell lines were both cultivated in a 2D standard cell culture (flasks, PAA, Germany) and a 3D 96 well plate hanging drop system (GravityPlusTM, Insphero, Switzerland) for 5 and 10 days, respectively. 3D microtissues were cultured either in mono- or in co-cultures with the human lung fibroblast cell line SV80. RNA expression profiling via Affymetrix chip analyses was performed with 3 biological replicates per cell line (A549, Colo699 and SV80) in mono- and co-cultures. For this purpose, RNA was isolated by a TriReagent standard protocol.
Cytotoxicity was determined for cisplatin and vinorelbine after 24 hours of incubation by ATP measurements of lysed microtissues/cells and modified AnnexinV/Propidium iodide staining for flow cytometry on Colo699 cells. Furthermore, standard flow cytometry cell cycle analyses and microscopic volume measurements were performed to analyse drug induced inhibition of proliferation.
Results: RNA expression profiles revealed within the A549 monocultures 892 and the Colo699 monocultures 1,042 regulated genes (≥ two-fold change in expression) for 2D versus 3D cultures, respectively. Between the cell lines A549 and Colo699 only a limited overlap (131 genes) in differentially expressed genes was detected. However, significant differences in gene expression were experienced in co-cultures with SV80. The most frequently altered genes are involved in the regulation of the cell cycle, organelle fission, RNA polymerases, histone methylation, DNA repair, oxidative stress and WNT signalling pathways. To prove the potential in-vivo relevance of these findings, we assessed the chemosensitivity in the Colo699 for 2D and 3D cell cultures. A significant difference in LD50 could be observed for cisplatin between 2D (100μM) and 3D (250μM) cultures in both ATP release measurements and AnnexinV/PI flow cytometry analyses. In cell cycle analyses the maximum M arrest for vinorelbine was achieved with 25μM in 2D, whereas in 3D 100μM of vinorelbine were needed. Cytotoxicity assays did not reveal a significant difference for vinorelbine in 2D versus 3D models.
Conclusion: Herein, we demonstrate that cultivation of cell lines either in a 2D or 3D environment lead not only to a difference in RNA expression but also in chemosensitivity. Thereby, these data support the urgent need of more in vivo like cell culture models for analysing drug related modes of action.
Citation Format: Gabriele Gamerith, Johannes Rainer, Arno Amann, Stefan Koeck, Edith Lorenz, Heinz Zwierzina, Julia M. Huber. Differences in RNA expression and chemosensitivity in 2D versus 3D non-small-cell lung cancer cultures. [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 326. doi:10.1158/1538-7445.AM2015-326