3D multicellular tumor spheroids (3D-MCTS) that closely mimic in vitro the complex lung tumor microenvironment (TME) are highly desirable for screening innovative anti-cancer therapeutics. Despite significant improvements in mimicking lung TME, few models have combined tumor-infiltrating mesenchymal stem cells from bone marrow (hBM-MSCs) with heterotypic 3D tumor spheroid models containing ECM mimetic components. Herein, we engineered hybrid 3D-MCTS that combine, for the first time, A549:fibroblasts:hBM-MSCs in heterotypic tri-culture, with bioinstructive hyaluronan microparticles that act as tumor-ECM mimetics and as cell-anchoring hotspots. The obtained results indicated that 3D microspheres provided proper support for cells to self-assemble into compact 3D microtissues and promoted an increase in CD44 expression, emulating the presence of native-ECM hyaluronan. 3D-MCTS size and sphere-like morphology was reproducible and tri-culture models presented the characteristic solid tumors necrotic core. Mesenchymal stem cells tracking in demonstrated that hBM-MSCs migrate to different regions in 3D microtumors mass exhibiting dynamic interactions with cancer cells and stromal fibroblasts, alike in human tumors. Importantly, doxorubicin administration revealed hBM-MSCs effect on cytotoxic responses in 3D tri-culture models and in dual cultures of hBM-MSCs:A549 at 10:1 ratio. Such findings evidence the relevance of including hBM-MSCs in combination with cancer-stromal fibroblasts in 3D in vitro tumor models and the importance to test different cell-to-cell ratios to mimic tumor heterogeneity. In addition, bioinstructive hyaluronan-microparticles were also effective as cell-agglomerating scaffolds and showed potential to be used as an enabling technology for including different ECM components in 3D in vitro models in the future.

在体外紧密模拟的3D多细胞肿瘤球体（3D-MCTS）复杂的肺肿瘤微环境（TME）对于筛选创新的抗癌治疗药物是非常理想的。尽管在模仿肺TME方面有显着改善，但很少有模型将来自骨髓的肿瘤浸润间充质干细胞（hBM-MSC）与包含ECM模拟成分的异型3D肿瘤球体模型相结合。在这里，我们设计了杂交3D-MCTS，它首次将A549：成纤维细胞：hBM-MSC在异型三培养中与具有生物指导意义的透明质酸微粒结合起来，所述微粒充当了肿瘤ECM模拟物和细胞锚定热点。获得的结果表明3D微球为细胞自组装成紧凑的3D微组织提供了适当的支持，并促进了CD44表达的增加，从而模拟了天然ECM透明质酸的存在。3D-MCTS大小和球状形态是可重现的，三培养模型显示了特征性实体瘤坏死核心。追踪间充质干细胞表明，hBM-MSCs迁移至3D微型肿瘤块中的不同区域，与人类肿瘤中的癌细胞和基质成纤维细胞表现出动态相互作用。重要的是，在3D三培养模型和hBM-MSCs：A549双重培养物中，阿霉素的给药显示了hBM-MSC对细胞毒性反应的作用，比例为10：1。这些发现证明了在3D模式下将hBM-MSC与癌症基质成纤维细胞结合使用的相关性 追踪间充质干细胞表明，hBM-MSCs迁移至3D微型肿瘤块中的不同区域，与人类肿瘤中的癌细胞和基质成纤维细胞表现出动态相互作用。重要的是，在3D三培养模型和hBM-MSCs：A549双重培养物中，阿霉素的给药显示了hBM-MSC对细胞毒性反应的作用，比例为10：1。这些发现证明了在3D模式下将hBM-MSC与癌症基质成纤维细胞结合使用的相关性 追踪间充质干细胞表明，hBM-MSCs迁移至3D微型肿瘤块中的不同区域，与人类肿瘤中的癌细胞和基质成纤维细胞表现出动态相互作用。重要的是，在3D三培养模型和hBM-MSCs：A549双重培养物中，阿霉素的给药显示了hBM-MSC对细胞毒性反应的作用，比例为10：1。这些发现证明了在3D模式下将hBM-MSC与癌症基质成纤维细胞结合使用的相关性体外肿瘤模型以及测试不同细胞比以模仿肿瘤异质性的重要性。此外，具有生物指导意义的透明质酸微粒还可以有效地作为细胞聚集的支架，并显示出将来有可能用作将不同的ECM组件包括在3D体外模型中的使能技术。