Abstract 1889: Formation and merger of H69 and H69AR small cell lung cancer (SCLC) tumor cell clusters (TCCs) in vitro
Authors: Ruprecht Nico O. , Hungerbuehler Martin , Kellmann Philipp , Harder Nathalie , Schmidt Günter , Wotzkow Carlos , Haenni Beat , Blank Fabian , Tengg-Kobligk Hendrik Von , Granzow Christof 
Published: July 2017

Abstract
A novel manifestation of tumor cell invasiveness, termed mutual cellular pervasion (MCP), was observed in vitro. It involved TCCs formed by SCLC cells.
Suspension cultures of H69 and H69AR cells were grown in flavin-free RPMI 1640 medium with 10% fetal bovine serum as the only source of flavins. Experiments and subcultivations were performed under light with wavelengths above 520 nm. TCC morphology was studied by confocal microscopy and transmission and scanning electron microscopy. Interactions of TCCs were followed by video microscopy in phase contrast and fluorescence modes. Data were analyzed using the Definiens software suite. The interaction of H69AR TCCs with confluent monolayers of human umbilical vascular endothelial cells (HUVEC) was visualized.
Freshly subcultivated, singularized tumor cells rapidly formed aggregates. After two days, solid TCCs prevailed whose surface cells were connected by desmosome-like junctions. Apoptosis but not necrosis became obvious in central parts of clusters with diameters above 100 to 150 μm. TCCs enlarged through both cell proliferation and serial cluster mergers. In the course of TCC merger, four stages were distinguishable: (1) directed cluster movement leading to contact of clusters, (2) formation of a near circular, symmetric structure termed contact disc. It consists of planar surface areas to which both TCCs involved contribute equally. (3) Merger of TCCs took place in the contact disc, and was accompanied by active movement of adjacent cells. (4) Starting from the merger zone, major surface areas of newly formed clusters were mutually pervaded by cells from both participating cell clusters. After numerous merger events, only low numbers of TCCs reaching sizes of up to 2 to 3 mm were present at subcultivation.
No spontaneous bursting, shrinkage, degeneration, or decay of TCCs has been observed. However, when TCCs of H69AR cells were confronted with confluent layers of HUVEC, drastic changes occurred in both formations. First, intense oscillatory cellular movements were triggered on TCC surfaces, preceding the release of cell aggregates which moved away from TCCs to the HUVEC layer. At the same time, HUVEC were disconnected from each other and started moving either randomly or in an oscillatory fashion. Individual tumor cells attached to the much larger HUVEC and spread on their surface, resulting in full covering which secluded HUVEC from the medium.
To our knowledge, neither formation nor merger of malignant TCCs in vitro have been described so far. TCC merger involves mutual cellular penetration of the involved TCC surfaces, i. e. MCP. Contrary to the invasion of tumor cells into non-tumor tissue, pervasion does not lead to destruction but results in TCC growth. However, key features of tumor cell invasion in vivo, such as chemotaxis, spreading and TCC formation as well as disruption of endothelial layers, manifest under conditions in vitro, thus facilitating related experimental studies.
Citation Format: Nico O. Ruprecht, Martin Hungerbuehler, Philipp Kellmann, Nathalie Harder, Günter Schmidt, Carlos Wotzkow, Beat Haenni, Fabian Blank, Hendrik Von Tengg-Kobligk, Christof Granzow. Formation and merger of H69 and H69AR small cell lung cancer (SCLC) tumor cell clusters (TCCs) in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1889. doi:10.1158/1538-7445.AM2017-1889