Data_Sheet_1_Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells.pdf

Endothelial cells (ECs) are an important component of the tumor microenvironment, playing key roles in tumor development and progression that span from angiogenesis to immune regulation and drug resistance. Heterotypic tumor spheroids are one of the most widely used in vitro tumor microenvironment models, presenting improved recapitulation of tumor microenvironments compared to 2D cultures, in a simple and low-cost setup. Heterotypic tumor spheroid models incorporating endothelial cells have been proposed but present multiple limitations, such as the short culture duration typically obtained, the use of animal-derived matrices, and poor reproducibility; the diversity of culture conditions employed hinders comparison between studies and standardization of relevant culture parameters. Herein, we developed long-term cultures of triple heterotypic spheroids composed of the HCC1954 tumor cell line, human fibroblasts, and ECs. We explored culture parameters potentially relevant for EC maintenance, such as tumor cell line, seeding cell number, cell ratio, and agitation vs. static culture. In HCC1954-based spheroids, we observed maintenance of viable EC for up to 1 month of culture in agitation, with retention of the identity markers CD31 and von Willebrand factor. At the optimized tumor cell:fibroblast:EC ratio of 1:3:10, HCC1954-based spheroids had a higher EC area/total spheroid area at 1 month of culture than the other cell ratios tested. EC maintenance was tumor cell line-dependent, and in HCC1954-based spheroids it was also dependent on the presence of fibroblasts and agitation. Moreover, vascular endothelial growth factor (VEGF) supplementation was not required for maintenance of EC, as the factor was endogenously produced. ECs co-localized with fibroblasts, which accumulated preferentially in the core of the spheroids and secreted EC-relevant extracellular matrix proteins, such as collagen I and IV. This simple model setup does not rely on artificial or animal-derived scaffolds and can serve as a useful tool to explore the culture parameters influencing heterotypic spheroids, contributing to model standardization, as well as to explore molecular cross talk of ECs within the tumor microenvironment, and potentially its effects on drug response.

内皮细胞（Endothelial cells, ECs）是肿瘤微环境（tumor microenvironment）的重要组成部分，在肿瘤发生与发展进程中发挥关键作用，涵盖从血管生成到免疫调控再到药物耐受的多个环节。异型肿瘤球体（heterotypic tumor spheroids）是目前应用最广泛的体外肿瘤微环境模型之一，相较于二维（2D）单层培养体系，该模型可更高效地重现肿瘤微环境的真实特征，且构建方案简便、成本低廉。尽管整合内皮细胞的异型肿瘤球体模型已被提出，但此类模型仍存在诸多局限：通常培养周期较短、需使用动物源性基质，且实验重复性较差；同时不同研究采用的培养条件差异显著，阻碍了相关研究间的横向对比与培养参数的标准化推广。
本研究构建了由HCC1954肿瘤细胞系、人成纤维细胞及内皮细胞组成的三重异型球体长期培养体系。我们系统探究了可能影响内皮细胞维持的多项培养参数，包括肿瘤细胞系选择、接种细胞总量、细胞接种比例，以及振荡培养与静态培养两种模式的差异。在以HCC1954细胞为基础构建的球体中，我们观察到：在振荡培养条件下，内皮细胞可维持存活状态长达1个月，且仍保留其标志性分子CD31与血管性血友病因子（von Willebrand factor）的表达。当采用优化后的细胞接种比例（肿瘤细胞:成纤维细胞:内皮细胞=1:3:10）时，培养至1个月的HCC1954来源球体中，内皮细胞面积占球体总面积的比例显著高于其他测试比例组。
内皮细胞的维持状态具有肿瘤细胞系依赖性，在以HCC1954为基础构建的球体中，该维持过程同时依赖于成纤维细胞的存在与振荡培养条件。此外，维持内皮细胞存活无需额外添加血管内皮生长因子（vascular endothelial growth factor, VEGF），因为该体系可内源产生该细胞因子。内皮细胞与成纤维细胞呈现共定位特征，而成纤维细胞优先聚集于球体核心，并分泌与内皮细胞功能相关的细胞外基质蛋白，如I型胶原蛋白与IV型胶原蛋白。
本模型构建流程简便，无需依赖人工或动物源性支架，可作为探究影响异型肿瘤球体培养参数的实用工具，有助于推动该类模型的标准化，同时可用于研究肿瘤微环境中内皮细胞的分子互作机制，及其对药物响应的潜在影响。