Metastable Intermediates Identified in Epithelial to Mesenchymal Transition are Regulated by G-quadruplex DNA Structures

Cancer is a heterogenous disease, with multiple cellular subpopulations present within a single tumour mass that differ genetically and morphologically, and thus respond differently to chemotherapeutics. Epithelial-to-Mesenchymal transition (EMT) has been shown to play a role in tumour heterogeneity. Single-cell sequencing is critical to identify cell-type-specific transcriptomic differences with multiplexing methods increasing experimental scope with reduced cost. Cell hashing with barcoded antibodies is commonly used to multiplex samples but is limited to samples expressing target antigens. Antigen-independent methods of barcoding cells, such as barcoded lipid-anchors, have gained traction but present substantial populations that cannot be unambiguously demultiplexed. Herein we report a multiplexed single-cell transfection-enabled cell hashing sequencing (scTECH-seq) platform, which uses antigen-independent endocytic uptake to barcode cells, resulting in efficient, uniform barcoding with high cell recovery. We apply this methodology to identify distinct metastable cell states in human mammary cells undergoing EMT and show that stabilisation of G-quadruplex DNA has the potential to inhibit EMT. Overall design: We performed single-cell RNA-seq of human mammary epithelial cells to identify metastable intermediate cell states in TGF-ß induced epithelial-to-mesenchymal transition (EMT). We also treated cells with pyridostatin to investigate the role G-quadruplex structures play in EMT. We combined this with our new method scTECH-seq to multiplex samples and quantify transcripts in the presence and absence of pyridostatin in TGF-ß induced EMT. Please note that processed data generated from both [GEX] and [CBO] samples, is linked to the corresonding [GEX] sample records.

癌症是一类具有异质性的疾病，单个肿瘤肿块内存在多种细胞亚群，这些亚群在遗传与形态学特征上存在差异，因此对化疗药物的响应各不相同。上皮间质转化（Epithelial-to-Mesenchymal Transition, EMT）已被证实与肿瘤异质性密切相关。单细胞测序（Single-cell Sequencing）是识别细胞类型特异性转录组差异的核心技术，而多重化实验方法可在降低实验成本的同时拓展实验规模。基于条形码抗体的细胞哈希（Cell hashing）技术常被用于样本多重化，但该方法仅适用于表达目标抗原的样本。抗原非依赖性的细胞条形码编码方法（如条形码脂质锚定物（barcoded lipid-anchors））已逐渐获得关注，但这类方法会产生大量无法被精准解多重的细胞群。本研究报道了一种可实现多重化的单细胞转染辅助细胞哈希测序（single-cell transfection-enabled cell hashing sequencing, scTECH-seq）平台，该平台利用抗原非依赖性的内吞作用对细胞进行条形码标记，可实现高效、均一的编码效果，且细胞回收率较高。我们将该方法应用于经历上皮间质转化的人乳腺细胞，以识别不同的亚稳态细胞状态，并证实G-四链体DNA（G-quadruplex DNA）的稳定化具有抑制上皮间质转化的潜力。实验设计概述：我们对人乳腺上皮细胞开展单细胞RNA测序，以识别转化生长因子-β（Transforming Growth Factor-β, TGF-β）诱导的上皮间质转化过程中的亚稳态中间细胞状态。我们同时使用吡咯斯塔汀（pyridostatin）处理细胞，以探究G-四链体结构在上皮间质转化中发挥的作用。我们将该处理流程与本研究开发的scTECH-seq技术相结合，对样本进行多重化，并定量检测TGF-β诱导的上皮间质转化过程中，添加与不添加吡咯斯塔汀时的细胞转录本水平。请注意，由[GEX]与[CBO]样本生成的已处理数据，均关联至对应的[GEX]样本记录。