Computational identification of surface markers for isolating distinct subpopulations from heterogeneous cancer cell populations [3’-Tag RNAseq]. Computational identification of surface markers for isolating distinct subpopulations from heterogeneous cancer cell populations [3’-Tag RNAseq]

Intratumoral heterogeneity reduces treatment efficacy and complicates our understanding of tumor progression. There is a pressing need to understand how heterogeneous tumor cell subpopulations coexist within a tumor, yet biological systems to study these processes in vitro are limited. With the advent of single-cell RNA sequencing (scRNA-seq), it has been found that some cancer cell lines contain distinct subpopulations. These heterogeneous cell lines provide a unique opportunity to study coexistence and evolution between genetically similar cancer cell subpopulations in controlled experimental settings. Here, we present scEMD, a computational package that returns candidate surface makers maximally unique to transcriptomic subpopulations in scRNA-seq which may be used for FACS isolation. scEMD was experimentally validated using the MDA-MB-231 and MDA-MB-436 cell lines. ESAM and BST2/Tetherin were experimentally confirmed to identify and separate scRNA-seq cluster-matched subpopulations of MDA-MB-231 and MDA-MB-436 cells, respectively. Identification and enrichment of distinct subpopulations within cell line models using this computationally efficient and experimentally validated workflow paves the way for studies on the generation and maintenance of cellular heterogeneity in low-complexity in vitro systems. Overall design: tagSeq on 2 isolated populations from the MDA-MB-231 breast cancer cell line and 2 isolated populations from the MDA-MB-436 breast cancer cell line.

肿瘤内异质性（Intratumoral heterogeneity）会降低治疗效果，同时使我们对肿瘤进展的认知更为复杂。当前亟需阐明异质性肿瘤细胞亚群在肿瘤内的共存机制，但体外研究此类过程的生物模型十分有限。
随着单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）技术的问世，研究人员发现部分癌细胞系中存在独特的细胞亚群。这类具有异质性的细胞系，为在可控实验环境下探究遗传背景相似的肿瘤细胞亚群间的共存与演化提供了独特机遇。
本研究介绍了一款名为scEMD的计算工具包，其可从单细胞RNA测序数据中筛选出对转录组亚群具有最高特异性的候选表面标志物，可用于荧光激活细胞分选（fluorescence-activated cell sorting, FACS）的细胞分离。研究团队使用MDA-MB-231与MDA-MB-436细胞系对scEMD进行了实验验证。实验证实，ESAM与BST2/Tetherin可分别用于识别并分离MDA-MB-231与MDA-MB-436细胞系中匹配单细胞RNA测序聚类结果的细胞亚群。
通过这款计算高效且经实验验证的分析流程，实现对细胞系模型中独特细胞亚群的识别与富集，可为低复杂度体外系统中细胞异质性的产生与维持机制研究铺平道路。
整体实验设计：对MDA-MB-231乳腺癌细胞系的2个分离亚群与MDA-MB-436乳腺癌细胞系的2个分离亚群开展tagSeq测序。