Heterogeneity is a hallmark of cancer. The advent of single-cell technologies has helped uncover heterogeneity in a high-throughput manner in different cancers across varied contexts. Here we apply single-cell sequencing technologies to reveal inherent heterogeneity in assumptively monoclonal pancreatic cancer (PDAC) cell lines and patient-derived organoids (PDOs). Our findings reveal a high degree of both genomic and transcriptomic polyclonality in monolayer PDAC cell lines, custodial variation induced by growing apparently identical cell lines in different laboratories, and transcriptomic shifts in transitioning from 2D to 3D spheroid growth models. Our findings also call into question the validity of widely available immortalized, non-transformed pancreatic lines as contemporaneous “control” lines in experiments. We confirm these findings using a variety of independent assays, including but not limited to whole exome sequencing, single-cell copy number variation sequencing (scCNVseq), single-nuclei assay for transposase-accessible chromatin with sequencing, fluorescence in-situ hybridization, and single-cell RNA sequencing (scRNAseq). We map scRNA expression data to unique genomic clones identified by orthogonally-gathered scCNVseq data of these same PDAC cell lines. Further, while PDOs are known to reflect the cognate in vivo biology of the parental tumor, we identify transcriptomic shifts during ex vivo passage that might hamper their predictive abilities over time. The impact of these findings on rigor and reproducibility of experimental data generated using established preclinical PDAC models between and across laboratories is uncertain, but a matter of concern.

异质性是癌症的标志。单细胞技术的出现有助于以高通量方式揭示不同背景下不同癌症的异质性。在这里，我们应用单细胞测序技术来揭示假定的单克隆胰腺癌 (PDAC) 细胞系和患者来源的类器官 (PDO) 的内在异质性。我们的研究结果揭示了单层 PDAC 细胞系中基因组和转录组学的高度多克隆性、在不同实验室中生长明显相同的细胞系引起的保管变异，以及从 2D 到 3D 球体生长模型转变的转录组学变化。我们的研究结果还质疑广泛使用的永生化、未转化的胰腺系作为实验中同期“对照”系的有效性。我们使用各种独立的检测方法证实了这些发现，包括但不限于全外显子组测序、单细胞拷贝数变异测序 (scCNVseq)、转座酶可及染色质单核检测测序、荧光原位杂交和单细胞 RNA 测序 (scRNAseq)。我们将 scRNA 表达数据映射到由这些相同 PDAC 细胞系的正交收集的 scCNVseq 数据识别的独特基因组克隆。此外，虽然已知 PDO 反映了亲本肿瘤的同源体内生物学，但我们发现了离体传代过程中的转录组学变化，这可能会随着时间的推移阻碍它们的预测能力。这些发现对实验室之间和跨实验室使用已建立的临床前 PDAC 模型生成的实验数据的严谨性和可重复性的影响尚不确定，