Open-ST: High-resolution spatial transcriptomics in 3D

Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst. The following fresh-frozen tissues were analyzed using our spatial transcriptomics method "Open-ST": E13 mouse head (sagittal section, C57BL/6N), p60 mouse brain hemisphere (coronal section, C57BL/6N), primary human grade-2 HPV-negative laryngeal head and neck squamous cell carcinoma (HNSCC), and two cervical level three lymph nodes from the same HNSCC patient (one healthy, one metastatic). From the HNSCC sample 19 serial sections were sequenced.空间转录组学（Spatial transcriptomics, ST）技术可揭示组织发育、稳态维持及疾病发生背后的分子机制。然而当前仍亟需兼具易用性、高分辨率、成本效益优势且可支持三维扩展的研究方法。在此我们报道了Open-ST：一种基于测序技术的开源实验与计算资源，旨在解决上述难题，并实现二维与三维层面的组织分子结构研究。在小鼠脑组织中，Open-ST实现了亚细胞分辨率的转录本捕获，并重构了细胞类型组成。在原发性头颈肿瘤及患者配对的健康/转移性淋巴结样本中，Open-ST成功捕获了免疫、基质及肿瘤细胞群的空间分布多样性，该结果已通过成像型空间转录组技术（imaging-based ST）得到验证。肿瘤组织中，独特的细胞状态围绕细胞间通讯热点区域聚集，但在转移性淋巴结中并无此排布模式。值得注意的是，对转移性淋巴结的三维重构与多模态分析，揭示了二维层面无法观测到的空间连续结构，以及精准定位于三维肿瘤-淋巴结边界的潜在生物标志物。所有实验方案与软件代码均可通过以下链接获取：https://rajewsky-lab.github.io/openst。本研究采用Open-ST空间转录组技术分析了以下新鲜冷冻组织样本：孕13天小鼠头部（矢状切面，C57BL/6N品系）、60日龄小鼠大脑半球（冠状切面，C57BL/6N品系）、原发性人类二级HPV阴性喉源性头颈鳞状细胞癌（HNSCC），以及同一名头颈鳞状细胞癌（HNSCC）患者的两枚颈Ⅲ区淋巴结（一枚为健康淋巴结，一枚为转移性淋巴结）。该头颈鳞状细胞癌样本共获取19张连续切片并进行了测序。