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. Impact Statement Cell adhesion is inherently different in two dimensional (2D) compared to three dimensional (3D) culture; yet, most adhesion assays in academia and industry are still conducted in 2D because few simple, yet effective, adhesion models exist in 3D. Recently we developed a 3D in vitro coculture model to examine breast epithelial spheroid interactions with endothelial tubes. We now show that this 3D coculture model can effectively be used to interrogate and quantify drug-induced differences in breast epithelial cell adhesion that are unique to 3D cocultures. This 3D coculture adhesion model can furthermore be modified for use with other cell types to better predict drug effects on cell-vasculature adhesion.

中文翻译：

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用于量化乳腺上皮细胞与内皮细胞粘附的三维体外共培养模型。

三维 (3D) 体外培养模型更好地概括了组织微环境，因此可以提供更好的平台来评估对粘附细胞 - 细胞相互作用的治疗效果。本研究的目的是确定 AD-01，一种 FK506 结合蛋白的肽衍生物，据报道与粘附受体 CD44 结合，是否会导致乳腺上皮球体对内皮管状网络的粘附更大减少我们的 3D 共培养模型系统与二维 (2D) 培养相比。MCF10A、MCF10A-NeuN、MDA-MB-231 和 MCF7 乳腺上皮细胞用 AD-01 作为单细胞或球体进行预处理。首先测量了乳腺上皮细胞对二维组织培养基质的粘附，其次是球体形成（乳腺细胞 - 细胞粘附）和球体粘附到基质胶或内皮网络。最后，量化了 2D 和 3D 培养中乳腺上皮细胞中的 CD44 表达。我们的结果表明 AD-01 对球体形成的影响最大，特别是在乳腺癌细胞系中。AD-01 还抑制乳腺癌球体粘附到内皮网络并沿内皮网络迁移。当培养为 3D 球体时，不同的乳腺上皮细胞系表达更多的 CD44，但这并没有普遍转化为更高的蛋白质水平。这项研究表明，3D 共培养模型可以对细胞粘附、迁移和细胞间相互作用提供独特的见解，从而增强对基本生物学机制的理解。此外，这种 3D 共培养系统也可能代表一个更相关的测试平台，用于了解新治疗剂的作用机制。影响陈述 与三维 (3D) 培养相比，二维 (2D) 培养中的细胞粘附本质上是不同的；然而，学术界和工业界的大多数粘附分析仍然在 2D 中进行，因为 3D 中几乎没有简单但有效的粘附模型。最近我们开发了一个 3D 体外共培养模型来检查乳腺上皮球体与内皮管的相互作用。我们现在表明，这种 3D 共培养模型可以有效地用于询问和量化药物诱导的 3D 共培养特有的乳腺上皮细胞粘附差异。