Department of Biomedical Engineering, School of Engineering, Saint Louis University, St Louis, MO, USA.
Department of Pharmacology and Physiology, School of Medicine, Saint Louis University, St Louis, MO, USA.
Acta Biomater. 2023 Jun;163:400-414. doi: 10.1016/j.actbio.2022.05.048. Epub 2022 Jun 1.
Glioblastoma (GBM) is the deadliest brain tumor for which there is no cure. Bioengineered GBM models, such as hydrogel-encapsulated spheroids, that capture both cell-cell and cell-matrix interactions could facilitate testing of much needed therapies. Elucidation of specific microenvironment properties on spheroid responsiveness to therapeutics would enhance the usefulness of GBM models as predictive drug screening platforms. Here, GBM spheroids consisting of U87 or patient-derived GBM cells were encapsulated in soft (∼1 kPa), stiff (∼7 kPa), and dual-stiffness polyethylene glycol-based hydrogels, with GBM spheroids seeded at the stiffness interface. Spheroids were cultured for 7 days and examined for viability, size, invasion, laminin expression, hypoxia, proliferation, and response to the chemotherapeutic temozolomide (TMZ). We noted excellent cell viability in all hydrogels, and higher infiltration in soft compared to stiff hydrogels for U87 spheroids. In dual gels spheroids mostly infiltrated away from the stiffness interface with minimal crossing over it and some individual cell migration along the interface. U87 spheroids were equally responsive to TMZ in the soft and stiff hydrogels, but cell viability in the spheroid periphery was higher than the core for stiff hydrogels whereas the opposite was true for soft hydrogels. HIF1A expression was higher in the core of spheroids in the stiff hydrogels, while there was no difference in cell proliferation between spheroids in the stiff vs soft hydrogels. Patient-derived GBM spheroids did not show stiffness-dependent drug responses. U87 cells showed similar laminin expression in soft and stiff hydrogels with higher expression in the spheroid periphery compared to the core. Our results indicate that microenvironment stiffness needs to be considered in bioengineered GBM models including those designed for use in drug screening applications. STATEMENT OF SIGNIFICANCE: Recent work on tumor models engineered for use in drug screening has highlighted the potential of hydrogel-encapsulated spheroids as a simple, yet effective platform that show drug responses similar to native tumors. It has also been shown that substrate stiffness, in vivo and in vitro, affects cancer cell responses to drugs. This is particularly important for glioblastoma (GBM), the deadliest brain cancer, as GBM cells invade by following the stiffer brain structures such as white matter tracks and the perivascular niche. Invading cells have also been associated with higher resistance to chemotherapy. Here we developed GBM spheroid models using soft, stiff and dual-stiffness hydrogels to explore the connection between substrate stiffness, spheroid invasion and drug responsiveness in a controlled environment.
胶质母细胞瘤（GBM）是目前尚无治愈方法的最致命脑肿瘤。生物工程化的 GBM 模型，如包埋球的水凝胶，可捕获细胞-细胞和细胞-基质相互作用，有助于测试急需的治疗方法。阐明球体对治疗的响应的特定微环境特性将增强 GBM 模型作为预测药物筛选平台的有用性。在这里，U87 或患者来源的 GBM 细胞组成的 GBM 球体被封装在软（约 1kPa）、硬（约 7kPa）和双硬度聚乙二醇水凝胶中，GBM 球体被种植在刚度界面上。将球体培养 7 天，并检查其活力、大小、侵袭性、层粘连蛋白表达、缺氧、增殖以及对化疗药物替莫唑胺（TMZ）的反应。我们注意到所有水凝胶中的细胞活力都很好，与硬水凝胶相比，U87 球体在软水凝胶中的渗透程度更高。在双凝胶中，球体主要从刚度界面向外渗透，很少穿过界面，并且一些单个细胞沿着界面迁移。U87 球体在软水凝胶和硬水凝胶中对 TMZ 的反应相同，但硬水凝胶中球体边缘的细胞活力高于核心，而软水凝胶则相反。在硬水凝胶中，球体核心的 HIF1A 表达更高，而在硬水凝胶和软水凝胶中，球体的细胞增殖没有差异。患者来源的 GBM 球体没有表现出依赖于刚度的药物反应。U87 细胞在软水凝胶和硬水凝胶中表现出相似的层粘连蛋白表达，与核心相比，在球体边缘的表达更高。我们的结果表明，微环境刚度需要在包括用于药物筛选应用的生物工程 GBM 模型中得到考虑。意义声明：最近关于用于药物筛选的肿瘤模型的工作强调了水凝胶包埋球体作为一种简单而有效的平台的潜力，该平台显示出与天然肿瘤相似的药物反应。还表明，体内和体外的基质刚度会影响癌细胞对药物的反应。这对于胶质母细胞瘤（GBM）尤其重要，GBM 是最致命的脑癌，因为 GBM 细胞通过沿着白质轨道和血管周围巢等较硬的大脑结构浸润。侵袭性细胞也与更高的化疗耐药性有关。在这里，我们使用软、硬和双硬度水凝胶开发了 GBM 球体模型，以在受控环境中探索基质刚度、球体侵袭和药物反应之间的联系。
