Supplementary Material for: Three-Dimensional Primary Cell Culture: A Novel Preclinical Model for Pancreatic Neuroendocrine Tumors

Molecular mechanisms underlying the development and progression of pancreatic neuroendocrine tumors (PanNETs) are still insufficiently understood. Efficacy of currently approved PanNET therapies is limited. While novel treatment options are being developed, patient stratification permitting more personalized treatment selection in PanNET is yet not feasible since no predictive markers are established. The lack of representative in vitro and in vivo models as well as the rarity and heterogeneity of PanNET are prevailing reasons for this. In this study, we describe an in vitro 3-dimensional (3-D) human primary PanNET culture system as a novel preclinical model for more personalized therapy selection. We present a screening platform allowing multicenter sample collection and drug screening in 3-D cultures of human primary PanNET cells. We demonstrate that primary cells isolated from PanNET patients and cultured in vitro form islet-like tumoroids. Islet-like tumoroids retain a neuroendocrine phenotype and are viable for at least 2 weeks in culture with a high success rate (86%). Viability can be monitored continuously allowing for a per-well normalization. In a proof-of-concept study, islet-like tumoroids were screened with three clinically approved therapies for PanNET: sunitinib, everolimus and temozolomide. Islet-like tumoroids display varying in vitro response profiles to <i>distinct therapeutic regimes</i>. Treatment response of islet-like tumoroids differs also <i>between patient</i> samples. We believe that the presented human PanNET screening platform is suitable for personalized drug testing in a larger patient cohort, and a broader application will help in identifying novel markers predicting treatment response and in refining PanNET therapy.

胰腺神经内分泌肿瘤（Pancreatic Neuroendocrine Tumors, PanNETs）发生发展的分子机制目前仍未被充分阐明。当前获批的PanNET治疗方案疗效有限。尽管新型治疗手段正处于研发阶段，但由于尚未确立有效的预测性生物标志物，能够实现更个性化治疗选择的PanNET患者分层仍不可行。造成这一困境的主要原因包括缺乏具有代表性的体外（in vitro）及体内（in vivo）模型，以及PanNET本身的罕见性与异质性。本研究报道了一种体外三维（3-D）人源原发性PanNET培养体系，作为可用于优化个性化治疗选择的新型临床前模型。我们开发了一套筛选平台，可实现多中心样本采集，并对人源原发性PanNET细胞的三维培养物开展药物筛选。研究证实，从PanNET患者体内分离得到的原代细胞经体外培养后可形成胰岛样肿瘤类器官。此类类器官可维持神经内分泌表型，且在培养体系中可存活至少2周，建模成功率高达86%。其存活状态可被持续监测，从而实现每孔样本的归一化校正。在一项概念验证研究中，我们采用三种临床获批的PanNET治疗药物——舒尼替尼（sunitinib）、依维莫司（everolimus）与替莫唑胺（temozolomide）对胰岛样肿瘤类器官进行了药物筛选。结果显示，胰岛样肿瘤类器官对不同治疗方案呈现出各异的体外响应特征，且不同患者来源的样本之间的治疗响应也存在差异。我们认为，本次报道的人源PanNET筛选平台可适用于更大规模患者队列的个性化药物检测，其更广泛的应用将有助于识别预测治疗响应的新型生物标志物，并推动PanNET治疗方案的优化。