The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.
St Vincent's Clinical School, Faculty of Medicine UNSW, Sydney, NSW, Australia.
Genome Med. 2021 May 10;13(1):81. doi: 10.1186/s13073-021-00885-z.
High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms.
Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments.
Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing.
We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.
高通量单细胞 RNA 测序（scRNA-Seq）已成为探索复杂人类癌症中细胞异质性的强大工具。使用新鲜人手术组织进行 scRNA-Seq 研究在物流上具有挑战性，排除了对样本的组织病理学分类，并且限制了批量处理的能力。当整合患者数据集时，这种障碍通常会引入技术偏差，并增加实验成本。尽管以前已经探索了组织保存方法来解决这些问题，但尚未在复杂的人类组织（如实体瘤）和高通量 scRNA-Seq 平台上进行检验。
我们使用 Chromium 10X 平台，对来自三个原发性乳腺癌、两个原发性前列腺癌和一个皮肤黑色素瘤的新鲜和冷冻保存的重复样本，共测序了约 120000 个细胞。我们对每种条件下的细胞进行了详细分析，以评估冷冻保存对细胞异质性、细胞质量、聚类和基因本体论鉴定的影响。此外，我们对单个乳腺癌样本进行了单细胞免疫表型分析，该样本以实体组织碎片的形式冷冻保存。
新鲜组织中鉴定的肿瘤异质性在冷冻保存的复制品中得到了很大程度的保留。我们表明，从冷冻组织碎片或冷冻细胞悬浮液中制备的单细胞测序与从新鲜组织中制备的单细胞测序相当，冷冻细胞悬浮液与新鲜组织在基因表达上具有更高的相关性。我们表明，冷冻保存对下游分析（如生物途径富集）的结果几乎没有影响。对于一些肿瘤，与新鲜分析的组织相比，冷冻保存稍微增加了细胞应激标志物。此外，我们证明了冷冻保存整个细胞对于使用 CITE-Seq 检测细胞表面蛋白的优势，这是使用其他保存方法（如单核测序）无法实现的。
我们表明，人类癌症的可行冷冻保存为多组学分析提供了高质量的单细胞。我们的研究为未来的临床单细胞 RNA 测序研究指导了组织生物库的新实验设计。
