Development of a 3D angiogenesis model to study tumour – endothelial cell interactions and the effects of anti-angiogenic drugs

Abstract

The tumour microenvironment and tumour angiogenesis play a critical role in the development and therapy of many cancers, but in vitro models reflecting these circumstances are rare. In this study, we describe the development of a novel tri-culture model, using non-small cell lung cancer (NSCLC) cell lines (A549 and Colo699) in combination with a fibroblast cell line (SV 80) and two different endothelial cell lines in a hanging drop technology. Endothelial cells aggregated either in small colonies in Colo699 containing microtissues or in tube like structures mainly in the stromal compartment of microtissues containing A549. An up-regulation of hypoxia and vimentin, ASMA and a downregulation of E-cadherin were observed in co- and tri-cultures compared to monocultures. Furthermore, a morphological alteration of A549 tumour cells resembling “signet ring cells” was observed in tri-cultures. The secretion of proangiogenic growth factors like vascular endothelial growth factor (VEGF) was measured in supernatants. Inhibition of these proangiogenic factors by using antiangiogenic drugs (bevacizumab and nindetanib) led to a significant decrease in migration of endothelial cells into microtissues. We demonstrate that our method is a promising tool for the generation of multicellular tumour microtissues and reflects in vivo conditions closer than 2D cell culture.

Effects of microtubule-inhibiting small molecule and antibody-drug conjugate treatment on differentially-sized A431 squamous carcinoma spheroids

“…To then further develop even more accurate representations of the natural cellular environment, co-cultures or organ-on-a-chip systems could be incorporated alongside live cell imaging 41,42 . Advancements in spheroid design have already been described in the literature, including angiogenesis models where endothelial networks are introduced into tumor spheroids to mimic the role that endothelial cells play in the tumor microenvironment 43 . Vascularization of the microtumors in an in vitro system has been demonstrated and could represent a major advance in the setup for in vitro experimentation 44 .…”

3D-cultivation of NSCLC cell lines induce gene expression alterations of key cancer-associated pathways and mimicin-vivoconditions

“…By these co-cultures a denser spheroid structure and cell-cell interactions can be achieved. These play an important role in EMT and hypoxia [ 48 , 49 ].…”

New artery of knowledge: 3D models of angiogenesis

“…Spheroids can be used to study the physiological spatial growth of vessel structures, the cell-cell interactions, and as a platform for drug development and discovery (30). Hybrid spheroids, generated by co-culturing two or three types of cell, are useful models to study the interaction between endothelial and tumour/stroma cells in different in vitro models of cancer (56,57) or between endothelial and mural cells in angiogenesis/vasculogenesis. Analysis of endothelial cell/MSC spheroids showed that MSC participated in the formation and stabilisation of luminal tubular structures, similarly to pericyte-like cells (58,59).…”