Comparison of imaging based single-cell resolution spatial transcriptomics profiling platforms using formalin-fixed paraffin-embedded tumor samples [MERFISH]

Imaging-based spatial transcriptomics (ST) is evolving as a pivotal technology in studying tumor biology and associated microenvironments. However, the strengths of the commercially available ST platforms in studying spatial biology have not been systematically evaluated using rigorously controlled experiments. We use serial 5 m sections of formalin-fixed, paraffin-embedded surgically resected lung adenocarcinoma and pleural mesothelioma samples in tissue microarrays to compare the performance of the ST platforms (CosMx, MERFISH, and Xenium (uni/multi-modal)) in reference to bulk RNA sequencing, multiplex immunofluorescence, GeoMx, and hematoxylin and eosin staining data. In addition to an objective assessment of automatic cell segmentation and phenotyping, we perform a manual phenotyping evaluation to assess pathologically meaningful comparisons between ST platforms. Here, we show the intricate differences between the ST platforms, reveal the importance of parameters such probe design in determining the data quality, and suggest reliable workflows for accurate spatial profiling and molecular discovery. We generated 2 TMAs with lung adenocarcinoma and 1 TMA with mesothelioma samples. MERFISH assay was performed with FFPE TMA sections.

基于成像的空间转录组学（Imaging-based spatial transcriptomics, ST）正逐步成为研究肿瘤生物学及其相关微环境的关键技术。然而，当前商用ST平台在空间生物学研究中的优势尚未通过严格控制的实验进行系统性评估。本研究利用组织微阵列（tissue microarrays, TMA）中手术切除的福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded）肺腺癌与胸膜间皮瘤样本的连续5 μm切片，以批量RNA测序（bulk RNA sequencing）、多重免疫荧光（multiplex immunofluorescence）、GeoMx以及苏木精-伊红染色（hematoxylin and eosin staining）数据为参照，对比CosMx、MERFISH及Xenium（单/多模态）等ST平台的性能。除对自动细胞分割与表型分型进行客观评估外，本研究还开展了人工表型分型评估，以分析不同ST平台间具有病理学意义的差异对比。本研究揭示了各ST平台间存在的复杂差异，阐明了探针设计等参数对数据质量的决定性作用，并提出了可用于精准空间图谱分析与分子发现的可靠实验流程。本研究共构建2套包含肺腺癌样本的组织微阵列与1套包含胸膜间皮瘤样本的组织微阵列。本研究针对福尔马林固定石蜡包埋（FFPE）组织微阵列切片完成了MERFISH检测。