Molecular weight specific impact of soluble and immobilized hyaluronan on CD44 expressing melanoma cells in 3D collagen matrices.
Molecular weight specific impact of soluble and immobilized hyaluronan on CD44 expressing melanoma cells in 3D collagen matrices.
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DOI：
10.1016/j.actbio.2016.12.026
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发表时间：
2017-03
影响因子：
9.7
通讯作者：
Jiranuwat Sapudom;Franziska Ullm;Steve Martin;Liv Kalbitzer;J. Naab;S. Möller;M. Schnabelrauch
中科院分区：
文献类型：
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作者：
Jiranuwat Sapudom;Franziska Ullm;Steve Martin;Liv Kalbitzer;J. Naab;S. Möller;M. Schnabelrauch
透明质酸(HA)及其主要受体CD44参与调节肿瘤细胞的扩散和转移。由于缺乏合适的基质模型，CD44-HA相互作用对肿瘤细胞增殖和侵袭的直接相关性依赖于HA的分子质量和呈现形式，目前还不完全清楚。为了解决这个问题，我们重建了3D胶原(Coll I)基质，并用相对分子质量为30-50 kDa(低分子量；LMW-HA)和500-750 kDa(高分子量；HMW-HA)的HA对其进行了功能化。采用后修饰策略将透明质酸共价固定到重组的纤维状胶原蛋白I基质上，得到了没有改变的胶原蛋白I网络结构，并在数天内稳定地固定化。采用激光共聚焦扫描显微镜、胶体探针力光谱和阿尔新蓝定量分析等方法对功能化的Coll I基质的拓扑结构、力学性能和HA含量进行了表征。为了阐明透明质酸依赖的肿瘤细胞的行为，在体外细胞实验中使用了表达CD44受体和不表达CD44受体的BRO黑色素瘤细胞系。我们发现只有可溶性LMW-HA以CD44依赖的方式促进细胞增殖，而HMW-HA和固定化LMW-HA不能促进细胞增殖。此外，只有固定化的LMW-HA具有增强的细胞侵袭能力。这两个发现都与LMW-HA和CD44+细胞在单细胞黏附测量中使用软胶体力光谱定量的非常强烈和特殊的黏附相互作用有关。总之，我们的结果引入了一种体外生物材料模型，允许测试透明质酸在3D纤维基质中的呈递模式和分子量特异性，从而模拟肿瘤微环境的重要生物特征。透明质酸(HA)的分子量和呈递形式(结合与溶解)的重要性被深入讨论，作为肿瘤进展和炎症的关键调节因素。我们引入了3D纤维状胶原基质，具有定义的微结构和硬度，允许以可溶和结合的方式呈现特定分子形式的HA。用这种方法模拟肿瘤微环境的重要活体特征，我们发现只有低分子HA(LMW-HA)以可溶性形式促进黑色素瘤细胞系(BRO)的增殖，而它以结合形式促进细胞侵袭。在单细胞水平的定量胶体力光谱中，LMW-HA的分子量特异性被证实是CD44受体依赖的，并且与黏附配体-受体的相互作用有关。
Hyaluronan (HA) and its principal receptor CD44 are known to be involved in regulating tumor cell dissemination and metastasis. The direct correlation of CD44-HA interaction on proliferation and invasion of tumor cells in dependence on the molecular weight and the presentation form of HA is not fully understood because of lack of appropriate matrix models. To address this issue, we reconstituted 3D collagen (Coll I) matrices and functionalized them with HA of molecular weight of 30–50 kDa (low molecular weight; LMW-HA) and 500–750 kDa (high molecular weight; HMW-HA). A post-modification strategy was applied to covalently immobilize HA to reconstituted fibrillar Coll I matrices, resulting in a non-altered Coll I network microstructure and stable immobilization over days. Functionalized Coll I matrices were characterized regarding topological and mechanical characteristics as well as HA amount using confocal laser scanning microscopy, colloidal probe force spectroscopy and quantitative Alcian blue assay, respectively. To elucidate HA dependent tumor cell behavior, BRO melanoma cell lines with and without CD44 receptor expression were used forin vitrocell experiments. We demonstrated that only soluble LMW-HA promoted cell proliferation in a CD44 dependent manner, while HMW-HA and immobilized LMW-HA did not. Furthermore, an enhanced cell invasion was found only for immobilized LMW-HA. Both findings correlated with a very strong and specific adhesive interaction of LMW-HA and CD44+ cells quantified in single cell adhesion measurements using soft colloidal force spectroscopy. Overall, our results introduce anin vitrobiomaterials model allowing to test presentation mode and molecular weight specificity of HA in a 3D fibrillar matrix thus mimicking importantin vivofeatures of tumor microenvironments.Statement of SignificanceMolecular weight and presentation form (bound vs. soluble) of hyaluronan (HA) are intensively discussed as key regulators in tumor progression and inflammation. We introduce 3D fibrillar collagen matrices with defined microstructure and stiffness allowing the presentation of specific molecular weight forms of HA in soluble and bound manner. Mimicking in that way importantin vivofeatures of tumor microenvironments, we found that only low molecular weight HA (LMW-HA) in soluble form promoted proliferation of a melanoma cell line (BRO), while it enhanced cell invasion in bound form. The molecular weight specificity of LMW-HA was verified to be CD44 receptor dependent and was correlated to adhesive ligand-receptor interactions in quantitative colloidal force spectroscopy at single cell level.