Comparative profiling of metastatic 4T1- versus non-metastatic Py230-based mammary tumors in an intraductal model for triple-negative breast cancer.. Comparative profiling of metastatic 4T1- versus non-metastatic Py230-based mammary tumors in an intraductal model for triple-negative breast cancer.

Purpose: Compare the tumor outgrowth and immunology of an immunocompetent 4T1- and Py230-based intraductal model for triple-negative breast cancer (TNBC). Methods: BALB/c-derived 4T1 and C57BL/6-derived Py230 mammary tumor cells were side-by-side intraductally inoculated in lactating and syngeneic mice, 4T1 and Py230 primary tumors were subsequently resected at 1, 3 and 6 weeks (w) post-inoculation (p.i.), and RNA was isolated from the 4T1 and Py230 primary tumors using in house developed protocols. Results: The differentially expressed genes in the 4T1 versus Py230 primary tumor datasets at the 3 different time points were categorized into 28 hallmark gene sets using GSEA. Nine hallmarks could be related to tumor immunology and collectively showed a decreased difference in expression over time, although every immunology-related gene set remained most expressed in 4T1 primary tumors across all time points. Seven hallmarks could be related to cellular mitosis and tumor progression and were significantly upregulated at 1 w p.i. in 4T1 primary tumors, whereas at 3 w p.i. they became significantly upregulated in Py230 primary tumors, indicative for enhanced Py230 tumor proliferation. The hallmark epithelial-mesenchymal transition was significantly upregulated at 3 w p.i. in 4T1 compared to Py230 primary tumors, indicative for metastatic progression. Gene sets involved in adipose/stromal processes within the mammary gland were downregulated or weakly expressed at 1 w p.i., but became upregulated at 3 w p.i. in 4T1 compared to Py230 primary tumors, indicating that at the time 4T1 tumor cells were less proliferative compared to Py230 tumor cells, the surrounding stroma and fat tissue were more active in the 4T1- compared to the Py230-based intraductal model. The hallmark androgen response also showed a significantly upregulated expression in Py230 tumors at 3 and 6 w p.i., indicating that androgen signaling is important in invasive Py230 tumors. Conclusion: Our findings highlight an innovative 4T1- and Py230-based intraductal mouse model for TNBC with a different tumor outgrowth and associated tumor microenvironment. These differential models may broadly represent the clinically observed TNBC diversity and together provide a powerful tool to evaluate therapeutics. Overall design: RNA was isolated from 4T1 and Py230 primary tumors at 3 different time points (i.e. 1, 3 and 6 w p.i.) using the Rneasy Mini Kit (QiAgen, Valencia, CA, USA). All analyses were done in triplicate. In addition, Matrigel® controls were included for each intraductal model and time point, yielding a total of 36 samples. RNA sequencing was done in collaboration with Oxford Genomics using Illumina’s TruSeq chemistry and a HiSeq4000 (paired end, read length of 75 bp) using 2 lanes aiming for 10 million reads per sample.

研究目的：对比基于免疫健全小鼠的4T1与Py230导管内模型的肿瘤生长动力学与免疫学特征，该模型用于三阴性乳腺癌（triple-negative breast cancer, TNBC）研究。
研究方法：将来自BALB/c小鼠的4T1与来自C57BL/6小鼠的Py230乳腺肿瘤细胞，同步接种于泌乳且同基因背景的小鼠乳腺导管内；分别于接种后（post-inoculation, p.i.）1、3、6周切除4T1与Py230原发肿瘤，并采用实验室自研方案从原发肿瘤中提取总RNA。
研究结果：通过基因集富集分析（Gene Set Enrichment Analysis, GSEA），将3个不同时间点的4T1与Py230原发肿瘤转录组差异表达基因划分为28个特征基因集。其中9个特征基因集与肿瘤免疫学相关，整体而言其表达差异随时间推移逐渐缩小，但所有免疫相关基因集在各时间点的4T1原发肿瘤中均呈高表达。7个特征基因集与细胞有丝分裂及肿瘤进展相关：在接种后1周，4T1原发肿瘤中该类基因集显著上调；而在接种后3周，该类基因集在Py230原发肿瘤中显著上调，提示Py230肿瘤增殖能力增强。上皮间质转化（epithelial-mesenchymal transition, EMT）特征基因集在接种后3周的4T1原发肿瘤中较Py230显著上调，提示存在转移性进展。乳腺内脂肪/基质相关基因集在接种后1周呈下调或低表达，但在接种后3周的4T1原发肿瘤中较Py230显著上调，表明当4T1肿瘤细胞增殖能力弱于Py230时，其周围基质与脂肪组织的活性高于Py230导管内模型。雄激素应答特征基因集在接种后3、6周的Py230肿瘤中显著上调，提示雄激素信号通路在侵袭性Py230肿瘤中发挥重要作用。
研究结论：本研究证实，基于4T1与Py230细胞系构建的导管内小鼠TNBC模型为创新型研究模型，二者具有截然不同的肿瘤生长特征与肿瘤微环境。该差异化模型可较好复现临床中观察到的TNBC异质性，共同为抗肿瘤治疗评估提供高效研究工具。
整体实验设计：采用RNeasy Mini试剂盒（Qiagen, 美国加利福尼亚州巴伦西亚）提取接种后1、3、6周的4T1与Py230原发肿瘤总RNA，所有实验均设置3次生物学重复。此外，为每个导管内模型与时间点设置基质胶（Matrigel®）对照组，最终共计36个样本。RNA测序工作由牛津基因组学中心（Oxford Genomics）协作完成，采用Illumina TruSeq建库试剂盒与HiSeq4000测序平台（双端测序，读长75 bp），使用2个测序泳道，目标测序深度为每个样本1000万条reads。