A Three-Dimensional In Vitro Coculture Model to Quantify Breast Epithelial Cell Adhesion to Endothelial Cells

Abstract

Three-dimensional (3D) in vitro culture models better recapitulate the tissue microenvironment, and therefore may provide a better platform to evaluate therapeutic effects on adhesive cell-cell interactions. The objective of this study was to determine if AD-01, a peptide derivative of FK506-binding protein like that is reported to bind to the adhesion receptor CD44, would induce a greater reduction in breast epithelial spheroid adhesion to endothelial tube-like networks in our 3D coculture model system compared to two-dimensional (2D) culture. MCF10A, MCF10A-NeuN, MDA-MB-231, and MCF7 breast epithelial cells were pretreated with AD-01 either as single cells or as spheroids. Breast epithelial cell adhesion to 2D tissue culture substrates was first measured, followed by spheroid formation (breast cell-cell adhesion) and spheroid adhesion to Matrigel or endothelial networks. Finally, CD44 expression was quantified in breast epithelial cells in 2D and 3D culture. Our results show that AD-01 had the largest effect on spheroid formation, specifically in breast cancer cell lines. AD-01 also inhibited breast cancer spheroid adhesion to and migration along endothelial networks. The different breast epithelial cell lines expressed more CD44 when cultured as 3D spheroids, but this did not universally translate into higher protein levels. This study shows that 3D coculture models can enable unique insights into cell adhesion, migration, and cell-cell interactions, thereby enhancing understanding of basic biological mechanisms. Furthermore, such 3D coculture systems may also represent a more relevant testing platform for understanding the mechanism-of-action of new therapeutic agents.

A first-in-human Phase I dose-escalation trial of the novel therapeutic peptide, ALM201, demonstrates a favourable safety profile in unselected patients with ovarian cancer and other advanced solid tumours

“…To better understand the underlying biological effects of the peptide, recent work using a 3D co-culture model of endothelial tubule growth and cancer cell migration suggests that ALM201, like its sister peptide AD-01, could have a direct impact on the attachment and migration of clusters of cancer cells along branched endothelial tubule networks [ 24 ]. This implies that ALM201 might also potentially interfere with the migration of cancer cells along blood vessels, thereby disrupting the complex microenvironment of solid tumours.…”

“…Interestingly, the inhibitory effect of the peptide was observed to be most striking in spheroids that over-expressed ERBB2 ( HER2/neu ), which suggests that tumours overexpressing HER2 might be more amenable to therapeutic intervention. In addition, in these experiments, transcripts of CD44 were increased when the cells were cultured as spheroids, but this did not appear to translate to increased levels of CD44 protein [ 24 ]. Whether or not these findings have any bearing on patients remains to be investigated.…”

Clustering human dental pulp fibroblasts spontaneously activate NLRP3 and AIM2 inflammasomes and induce IL-1β secretion

“…Thus, for better illustrating the regulation mechanism, it is valuable to find an in vitro research model that simulates aseptic pulpitis. The tissue microenvironment can be recapitulate using three-dimensional culture model to provide good platform for investigation of pulpitis pathogenesis [ 27 ]. Nemosis is a newly discovered way of fibroblast activation.…”

Successful Proof-of-Concept for Topical Delivery of Novel Peptide ALM201 with Potential Usefulness for Treating Neovascular Eye Disorders

“…This may relate directly to the putative mechanism of action of ALM201, where its effects on endothelial cell biology and vessel integrity would be important in terms of upstream pathologic features to target at an early stage of nAMD. 35 Alternatively, it may reflect a limitation of the dose, duration of dosing, or limitations of the model itself (i.e., induction of neovascular lesions with a laser as opposed to de novo disease). Regardless, to achieve this level of efficacy with daily administrations of low micromolar doses in a simple, nonoptimized formulation provides a very strong basis on which to develop ALM201 topically further for eye conditions.…”