Supplementary Material for: Co-culture device for in vitro high throughput analysis of cancer associated fibroblast and cancer cell interactions

Abstract Introduction Cancers in general, and specifically lung cancer, continue to have low patient survival rates when the patient is at an advanced stage when diagnosed. It appears that the local environment, especially fibroblasts and their signaling molecules, tends to induce metastasis, increase cancer cell resistance to treatment, and aid in tumor growth rates. Since 3-D models quickly become too complex and/or expensive, and therefore rarely leave the lab they are developed in, it is interesting to develop a 2-D model that more closely mimics the clustered tumor formation and bulk interaction with a surrounding fibroblast environment. Methods In the present study, we utilize an off-the-shelf stereolithography 3-D printer, standard use well plates, magnets, and metallic beads to create a customizable 2-D co-culture system capable of being analyzed quantitatively with staining and qualitatively with standard fluorescent/brightfield microscopy to determine cancer-fibroblast interactions while also being able to test chemotherapeutic drugs in a high-throughput manner with standard 96-well plates. Results Comparisons from monoculture and co-culture growth rates shows that the presence of fibroblasts allows for significantly increased growth rates for H460 cancer. Additionally, viability of cancer cells can be quantified with simple cell staining methods and morphology and cell-cell interactions can be observed and studied. Discussion The high throughput model demonstrates that boundary condition changes can be observed between cancer cells and fibroblasts based upon the different chemotherapeutics that have been administered.

摘要 引言
总体而言，各类癌症（尤以肺癌为甚）在患者确诊时已处于晚期的情况下，患者生存率仍处于低位。研究表明，肿瘤局部微环境——尤其是成纤维细胞及其信号分子——可诱导肿瘤转移、增强癌细胞的治疗抗性并促进肿瘤生长。由于三维（3-D）模型往往会迅速变得过于复杂且成本高昂，因此极少能脱离其开发的实验室环境，故开发一种能够更精准模拟肿瘤簇形成以及与周围成纤维细胞环境整体互作的二维（2-D）模型具有重要研究价值。
方法
本研究采用市售立体光刻3D打印机（stereolithography 3-D printer）、标准多孔板、磁体及金属微珠，构建了一种可定制的二维共培养系统（co-culture system）。该系统可通过染色法实现定量分析，通过标准荧光/明场显微镜开展定性观察，用以探究癌细胞与成纤维细胞的互作关系；同时可依托标准96孔板以高通量方式进行化疗药物测试。
结果
单培养（monoculture）与共培养的生长速率对比结果显示，成纤维细胞的存在可显著提升H460癌细胞的生长速率。此外，可通过简便的细胞染色方法对癌细胞活力进行定量检测，还可观察并研究细胞形态与细胞间互作情况。
讨论
本高通量模型表明，基于施用的不同化疗药物，可观察到癌细胞与成纤维细胞之间的边界条件变化。