Abstract 2017: Development of a novel 3D tri-culture system in an in vitro non-small cell lung cancer (NSCLC) model

Abstract

Abstract Introduction: Different molecular processes lead to metastatic spread and the occurrence of tumor cell resistance to therapeutic interventions. Among them, the stromal compartment of tumors plays a key role. We describe the development of our 3D co-culture to a novel tri-culture model, using non-small cell lung cancer (NSCLC) cell lines in combination with a lung fibroblast and two different endothelial cell lines in a hanging drop technology. Methods: 96-well hanging drop microtiter plates (InSphero AG, Zürich, Switzerland) were applied for the production of 3D mono-, co- and tri-cultures including the human lung cancer cell lines A549 or Colo699 alone or in combination with a human lung fibroblast cell line (SV-80) and either the human umbilical vein endothelial cell line (HUVEC) or the human lung microvascular endothelial cell line (HMVEC-L). In addition, to conventional histology (H&E, PAS) tumor fibroblast spheroid aggregation was displayed immunohistochemically (IHC) by protein expression of e-cadherin, vimentin, Ki67, CD31 and α-smooth muscle actin (α-SMA). Viability of each cell compartment of the microtissue was determined by a modified AnnexinV/Propidium Iodide staining for flow cytometry. Results: Endothelial cells aggregated either in small colonies with Colo699 or as single cells mainly in the stromal compartment of A540 microtissues. Simultaneously an up-regulation of vimentin and a downregulation of E-Cadherin was observed in co- and tri-cultures compared to monocultures. Furthermore, Ki67 expression increased significantly from mono- to co- and tri-cultures in both cancer cell lines, indicating a high metabolic activity. In addition, a morphological alteration of A549 tumor cells resembling “signet ring cells” was observed for the first time in both endothelial tri-cultures with a significantly increased glycoprotein expression demonstrated by a Periodic acid-Schiff (PAS) stain. Conclusion: We demonstrate that our method is a promising tool for the generation of multicellular tumor microtissues and reflects in vivo conditions closer than traditional 2D cell culture. Furthermore, it represents an appropriate alternative method to investigate endothelial cell interactions with tumor and stromal cells. Citation Format: Arno Amann, Marit Zwierzina, Julia M. Huber, Gabriele Gamerith, Mario Bitsche, Elisabeth J. Pechriggl, Wolfgang Hilbe, Heinz Zwierzina. Development of a novel 3D tri-culture system in an in vitro non-small cell lung cancer (NSCLC) model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2017. doi:10.1158/1538-7445.AM2014-2017