Characterization of tumor cell lines derived from genetically engineered mouse models of bladder cancer

We have generated various genetically engineered mouse models (GEMMs) of advanced bladder cancer that recapitulate various morphological variants of human tumors, with distinct histological characteristics, differing onset times or growth rates, and varying incidences of dissemination. To investigate the molecular pathways underlying these features, we performed a transcriptomic analysis of these models in comparison with control urothelium. Given that concurrent TP53 and RB1 alterations are frequently observed in human tumors and are associated with specific molecular subtypes and increased aggressiveness, this study aimed to develop and characterize new models comparing the combined loss of Pten, Trp53, and Rb1 (in an Rbl1-null background, due to functional compensation of Rb1 loss in mouse tissues) with the loss of Pten and Trp53 alone, where the retinoblastoma (Rb) family remains intact. In both models, basal urothelial cells were targeted using adenoviruses expressing Cre unther de control of the Krt5 promoter (Ad5-Krt5-Cre), while luminal urothelial cells were targeted using the murine Krt20 promoter (Ad5-Krt20-Cre). Despite the advantages offered by these GEMMs, their limitations—including unpredictable tumor onset, long latency periods, and difficulties in early tumor detection—led us to develop immunocompetent heterotopic models using tumor cell lines derived from primary tumors arising in GEMMs. These models enable more accurate tumor monitoring and synchronized growth, thereby improving the robustness of preclinical studies. Here, we provide a transcriptomic analysis of some of these bladder tumor-derived cells. Overall design: Gene expression was compared among bladder tumor-derived cells generated from injection of Cre-expressing adevoviruses into the bladder of conditional Quadruple Knock-Out (QKO; Ptenflox/flox, Trp53flox/flox, Rb1flox/flox, Rbl1-/-) and Double Knock-Out (DKO; Ptenflox/flox, Trp53flox/flox) mice. This transcriptomic study included 1 DKO-Krt5 tumor-derived cell line (4K5), 1 DKO-Krt20 tumor-derived cell line (3K20), 2 QKO-Krt5 tumor-derived cell lines (CK5.A and K5.6), and 1 QKO-Krt20 tumor-derived cell line (CK20.D). Each cell line was analyzed in duplicate.

本研究构建了多种晚期膀胱癌基因工程小鼠模型（genetically engineered mouse models, GEMMs），这些模型可重现人类肿瘤的多种形态学变异类型，具备独特的组织学特征，且肿瘤发生时间、生长速度各不相同，播散发生率亦存在差异。为探究这些表型背后的分子通路，本研究对这些模型与对照尿路上皮进行了转录组分析。鉴于人类肿瘤中常同时存在TP53与RB1突变，且该类突变与特定分子亚型及侵袭性增强相关，本研究旨在构建并表征新型模型，对比Pten、Trp53与Rb1联合缺失（由于小鼠组织中Rb1缺失存在功能代偿，故构建于Rbl1敲除背景中）与仅缺失Pten和Trp53（视网膜母细胞瘤（retinoblastoma, Rb）家族功能完整）的表型差异。在两种模型中，研究均通过以Krt5启动子驱动的腺病毒（Ad5-Krt5-Cre）靶向基底尿路上皮细胞，同时以小鼠Krt20启动子驱动的腺病毒（Ad5-Krt20-Cre）靶向管腔尿路上皮细胞。尽管此类基因工程小鼠模型具备诸多优势，但它们也存在局限性：肿瘤发生不可预测、潜伏期长且早期肿瘤检测难度大，为此我们利用源自基因工程小鼠模型原发肿瘤的肿瘤细胞系，构建了免疫健全的异位移植模型。该类模型可实现更精准的肿瘤监测与同步化生长，从而提升临床前研究的可靠性。本研究现已对部分此类膀胱肿瘤来源细胞开展转录组分析。整体实验设计：本研究对比了通过向条件性四重敲除（Quadruple Knock-Out, QKO；Ptenflox/flox, Trp53flox/flox, Rb1flox/flox, Rbl1-/-）与条件性双敲除（Double Knock-Out, DKO；Ptenflox/flox, Trp53flox/flox）小鼠膀胱内注射表达Cre的腺病毒所获得的膀胱肿瘤来源细胞的基因表达水平。本次转录组研究共纳入1株DKO-Krt5来源肿瘤细胞系（4K5）、1株DKO-Krt20来源肿瘤细胞系（3K20）、2株QKO-Krt5来源肿瘤细胞系（CK5.A与K5.6）以及1株QKO-Krt20来源肿瘤细胞系（CK20.D），每个细胞系均进行2次重复检测。