Culture media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids. Culture media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids

Spheroids, near spherical multicellular aggregates, are one of the most common types of threedimensional (3D) cell cultures. Despite decades of implementation of spheroid technology in various fields of life science and medical research, no minimal information (MI) guidelines are available to cope with heterogeneity and to stimulate transparency. To cope with this unmet need, we assembled an international consortium to develop the MISpheroID knowledgebase (https://www.mispheroid.org) and interrogation revealed heterogeneity and lack of transparency in published spheroid-related experiments. This steered us to empirically evaluate the impact of cell line, culture medium type, spheroid formation method and spheroid size on complementary spheroid metrics (RNA fingerprints, presence of cell death, ATP content, glucose to lactate conversion, secreted protein signatures, circularity, size and cancer therapy response). We measured media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids using RNA sequencing (RNA-seq). These spheroids were formed in ultra-low attachment plates and cultured in 6 different media types (DMEM high glucose, DMEM/F12, RPMI1640, DMEM low glucose, EMEM and MEM) for 5 (HCT116) or 7 (A549, SKOV3 and U87MG) days. RNA extraction was performed on 2 spheroids per condition using the miRNeasy micro kit (217084, Qiagen, Hilden, Germany). RNA-sequencing libraries were prepared from purified RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) according to the manufacturer's instructions, using 27.5ng of RNA that was DNase treated using HL-dsDNA (Arcticzymes, TromsØ, Norway). The individual libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Pleasanton, CA, US) and equimolarly pooled. The pool concentration was measured with Qubit and 1.4pM with 1% PhiX was sequenced on a NextSeq 500 (Illumina, San Diego, CA, US) using a high-output 1x75 run. Reads were mapped to the human genome using Tophat and gene expression counts were generated using HTSeq. This data was used to perform Principal Component Analysis (PCA) and Gene Set Enrichment Analysis (GSEA).

球体（Spheroids），即近球形多细胞聚集体，是最常见的三维（3D）细胞培养体系之一。尽管球体技术已在生命科学与医学研究的诸多领域应用数十年，但目前尚无针对异质性问题、旨在提升研究透明度的最小信息（Minimal Information, MI）指南。为填补这一未被满足的研究需求，我们组建了国际联盟以开发MISpheroID知识库（https://www.mispheroid.org），且经调研发现已发表的球体相关实验普遍存在异质性与透明度缺失的问题。这一发现促使我们通过实验评估细胞系、培养基类型、球体构建方法以及球体尺寸对一系列互补性球体表征指标的影响，这些指标包括RNA指纹图谱、细胞死亡情况、ATP含量、葡萄糖向乳酸的转化、分泌蛋白特征、圆度、尺寸以及癌症治疗响应性。我们通过RNA测序（RNA-seq）检测了肺癌（A549）、结直肠癌（HCT116）、卵巢癌（SKOV3）及胶质母细胞瘤（U87MG）的球体中由培养基诱导的转录组差异。这些球体均通过超低吸附培养板构建，并在6种不同培养基（高糖DMEM、DMEM/F12、RPMI1640、低糖DMEM、EMEM及MEM）中分别培养：HCT116细胞球体培养5天，A549、SKOV3及U87MG细胞球体培养7天。每个培养条件下取2个球体进行RNA提取，使用miRNeasy微型试剂盒（货号217084，凯杰公司，德国希尔德）。取27.5 ng经HL-dsDNA（Arcticzymes，挪威特罗姆瑟）进行DNase处理后的RNA，按照制造商说明书，使用适配Illumina平台的QuantSeq 3' mRNA测序文库制备正向试剂盒（Lexogen，奥地利维也纳）构建RNA测序文库。采用qPCR法结合KAPA文库定量试剂盒（罗氏，美国加州普莱森顿）对各文库进行定量，并将文库按等摩尔浓度混合。使用Qubit检测混合文库的浓度，随后以1.4 pM的文库浓度（添加1% PhiX对照），在NextSeq 500测序仪（Illumina，美国加州圣迭戈）上采用高产出1×75 bp测序模式进行测序。使用Tophat将测序读段比对至人类基因组，并用HTSeq生成基因表达计数矩阵。本数据集被用于开展主成分分析（Principal Component Analysis, PCA）与基因集富集分析（Gene Set Enrichment Analysis, GSEA）。