Multicellular spheroids as a three-dimensional tumor cell model for esophageal und esophagogastric junction cancers

Working with cell lines in monolayer culture implies a lot of difficulties regarding the comparability of the behaviour to the complex in vivo situation including growth and resistance mechanisms. Three-dimensional multicellular spheroids (MCS) supplied per diffusionem are known to mimic the growth characteristics of solid tumors and to develop gradients in nutriens like oxygen and degradation products with increasing size. Cells cultivated as MCS are higher differentiated than in monolayer culture and have a similar extracellular matrix system. The model simulates solid micrometastases and microregions between blood vessels and enables experiments concerning the microenvironment and formation of subpopulations. Aim of this study was to establish such a three-dimensional model for established cell lines derived from an esophageal squamous (OE21) and adenocarcinoma (OE33) as well as cardia cancer (OE19). Cell lines were purchased from the European Collection of Animal Cell Cultures, cultivated as spheroids in a spinner system using enriched RPMI 1640 medium. Growth behaviour and morphology were analysed. Lethal cells were detected by staining with SYTOX® and necrosis formation by Luzifer Yellow VS. In addition, the production of reactive oxygen species was compared between the three different sheroid entitites through DCF-staining. We report the successful establishment of MCS derived from established cell cultures of esophageal and esophagogastric junction carcinomas. Cell morphology was completely different depending on their tumor origin. The MCS displayed a higher differented morphology compared to the respective monolayer cultures (OE21: bulky spheroids within hours, 1000µm; OE19: round shaped, mucin-producing, 500µm within 1 week; OE33: small sheroids, 100µm). Lethal cells and nectrotic areas were predominantly detectable in OE21 sheroids. Reactive oxygen species were intensively detected in both adenocarcinoma sheroids derived from Barrett's cancer and cardia cancer compared to squamous cell cancer suggesting similarity to the in vivo situation. Within a short period of time tumor sheroids from esophageal and cardia cancer cell lines can be repeatedly produced for various biochemical and molecular studies.

Keywords: OE19, OE21, OE33, esophageal and esophagogastric junction cancers, multicellular tumor spheroids