Systematic comparison of pancreatic ductal adenocarcinoma models identifies a conserved highly plastic basal cell state

Pancreatic ductal adenocarcinoma (PDAC) portends a dire prognosis. Intra-tumoral heterogeneity and cellular plasticity have emerged as hallmarks of cancer, including PDAC. Yet, our understanding of the mechanisms underpinning cellular diversity in PDAC remains limited. Here, we investigate cellular heterogeneity of PDAC cancer cells across a range of in vitro and in vivo growth conditions using single-cell genomics. We find heterogeneity contracts significantly in 2D and 3D cell culture models but becomes restored upon orthotopic transplantation. Orthotopic transplants reproducibly acquire cell states identified in autochthonous PDAC tumors, including a basal state exhibiting co-expression and co-accessibility of epithelial and mesenchymal genes. Using linage-tracing combined with single-cell transcriptomics, we demonstrate basal cells display high plasticity in situ. This work defines the impact of cellular growth conditions on phenotypic diversity and uncovers a highly plastic cell state with the capacity to facilitate state transitions and promote intra-tumoral heterogeneity in PDAC. Overall design: Single-cell RNA-sequencing data of pancreatic ductal adenocarcinoma was generated on the 10X Chromium platform from either autochthonous KPCT mice or from orthotopic pancreas transplants. Autochthonous KPCT mice were generated by crossing previously published KrasLSL-G12D/+ , Trp53flox/flox , Pdx1-Cre, and Rosa26LSL-tdTomato mice. For Lgr5 co-expression studies, the KPCT mice above were crossed with Lgr5GFP-IRES-CreER/+ reporter mice. To generate various orthotopic PDAC transplant models we have implanted into pancreas of NSG recipient mice either: ~3mm PDAC fragments generated from KPCT primary tumors; or tumor cells derived from KPCT primary PDAC tumors.

胰腺导管腺癌（Pancreatic ductal adenocarcinoma, PDAC）的预后极差。瘤内异质性与细胞可塑性已成为包括胰腺导管腺癌在内的多种癌症的标志性特征。然而，目前学界对胰腺导管腺癌中细胞多样性背后的调控机制仍知之甚少。本研究借助单细胞基因组学技术，探究了不同体外（in vitro）与体内（in vivo）培养条件下胰腺导管腺癌细胞的异质性特征。研究发现，胰腺导管腺癌细胞的异质性在二维（2D）与三维（3D）细胞培养模型中显著收窄，但在原位移植（orthotopic transplantation）后可恢复至原有水平。原位移植模型可稳定重现自发形成的胰腺导管腺癌肿瘤中存在的细胞状态，其中包括一种同时表达上皮与间质基因、且二者染色质开放状态一致的基底样细胞状态。本研究结合谱系示踪（lineage-tracing）技术与单细胞转录组学技术，证实基底样细胞在原位（in situ）具有极高的可塑性。本研究明确了细胞培养条件对胰腺导管腺癌细胞表型多样性的影响，并揭示了一种具有高度可塑性的细胞状态：该状态可推动细胞状态转换，进而促进胰腺导管腺癌的瘤内异质性。

实验设计概述：本研究的胰腺导管腺癌单细胞RNA测序数据均基于10X Chromium平台生成，样本来源分为两类：自发形成胰腺导管腺癌的KPCT小鼠，以及胰腺原位移植模型。KPCT小鼠通过将已发表的KrasLSL-G12D/+、Trp53flox/flox、Pdx1-Cre及Rosa26LSL-tdTomato小鼠进行杂交构建获得。针对Lgr5共表达研究，本研究将上述KPCT小鼠与Lgr5GFP-IRES-CreER/+报告基因小鼠进行杂交。为构建多种原位胰腺导管腺癌移植模型，本研究将两类样本植入NSG免疫缺陷小鼠的胰腺中：一是从KPCT原代肿瘤分离得到的约3mm大小的胰腺导管腺癌组织块；二是从KPCT原代胰腺导管腺癌肿瘤中分离得到的肿瘤细胞。