Paper-Based Coculture Platform to Evaluate the Effects of Fibroblasts on Estrogen Signaling in ER+ Breast Cancers.
Paper-Based Coculture Platform to Evaluate the Effects of Fibroblasts on Estrogen Signaling in ER+ Breast Cancers.
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DOI：
10.1021/acsmeasuresciau.3c00032
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发表时间：
2023-12-20
期刊：
影响因子：
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通讯作者：
Lockett, Matthew R
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文献类型：
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作者：
Sitte, Zachary R;Miranda Buzetta, Abel Andre;Jones, Sarina J;Lin, Zhi-Wei;Whitman, Nathan Ashbrook;Lockett, Matthew R
基于细胞的检测能够在分子水平上研究细胞对候选药物或潜在毒素的反应。反式激活测定定量核受体的激活或抑制，核受体是哺乳动物细胞中基因靶点的关键转录调节因子。一种这样的测定将荧光素酶的表达与雌激素受体-α（ER α）的转录活性偶联。虽然该试验经常用于筛选雌激素信号传导途径的激动剂和拮抗剂，但该装置依赖于单层培养，其中细胞直接接种在细胞相容性塑料器皿的表面上。肿瘤微环境不仅仅是癌细胞的集合，并且受到组织结构、细胞外基质的存在和由非癌相邻细胞产生的细胞间信号传导分子（例如，成纤维细胞）。存在对三维培养平台的需要，所述三维培养平台可以快速原型化以评估新的构型，并且容易大量生产用于转化研究和筛选应用。在这里，我们展示了纸基培养平台的实用性，以探测两种细胞类型之间的细胞间信号传导的影响。我们使用纸支架来产生类似肿瘤的环境，形成一定体积的乳腺癌细胞悬浮在胶原蛋白中。通过将纸支架放置在商业96孔板中，我们比较了仅乳腺癌细胞的单培养物与在不同位置含有成纤维细胞的共培养物配置，其模拟了乳腺癌进展的阶段。我们发现T47D-KBluc细胞系中ER α的反式激活受成纤维细胞的存在、数量和接近程度的影响，并且是细胞间信号分子的结果。在筛选成纤维细胞分泌的信号分子的小文库后，我们发现白细胞介素-6（IL-6）是降低雌二醇敏感性的主要驱动因素。在共培养配置中，通过添加IL-6中和抗体减轻了这些影响。我们还评估了雌激素受体的表达和转录调控，进一步证明了纸基平台的详细机制研究的实用性。
Cell-based assays enable molecular-level studies of cellular responses to drug candidates or potential toxins. Transactivation assays quantify the activation or inhibition of nuclear receptors, key transcriptional regulators of gene targets in mamalian cells. One such assay couples the expression of luciferase to the transcriptional activity of estrogen receptor-alpha (ERα). While this assay is regularly used to screen for agonists and antagonists of the estrogen signaling pathway, the setup relies on monolayer cultures in which cells are plated directly onto the surface of cell-compatible plasticware. The tumor microenvironment is more than a collection of cancerous cells and is profoundly influenced by tissue architecture, the presence of extracellular matrices, and intercellular signaling molecules produced by non-cancerous neighboring cells (e.g., fibroblasts). There exists a need for three-dimensional culture platforms that can be rapidly prototyped to assess new configurations and readily produced in the large numbers needed for translational studies and screening applications. Here, we demonstrate the utility of the paper-based culture platform to probe the effects of intercellular signaling between two cell types. We used paper scaffolds to generate tumor-like environments, forming a defined volume of breast cancer cells suspended in collagen. By placing the paper scaffolds in commercial 96-well plates, we compared monocultures of only breast cancer cells with coculture configurations containing fibroblasts in different locations that mimicked the stages of breast cancer progression. We show that ERα transactivation in the T47D-KBluc cell line is affected by the presence, number, and proximity of fibroblasts, and is a consequence of intercellular signaling molecules. After screening a small library of fibroblast-secreted signaling molecules, we showed that interleukin-6 (IL-6) was the primary driver of reduced estradiol sensitivity. These effects were mitigated in the coculture configurations by the addition of an IL-6 neutralizing antibody. We also assessed estrogen receptor expression and transcriptional regulation, further demonstrating the utility of the paper-based platform for detailed mechanistic studies.