Analysis of differentiation therapeutic approaches on murine breast cancer organoids

In this experiment, we have tested the effect of microRNA-203 as a potential differentiation inducer in breast cancer organoids, derived from the PyMT mouse model. MMTV-PyVT transgenic mice express the Polyoma Virus middle T antigen under the direction of the mouse mammary tumor virus promoter/enhancer. Hemizygous MMTV-PyMT females develop palpable mammary tumors which metastasize to the lung. These mice have high penetrance of early onset of mammary cancer compared to other mammary tumor models. PyMT mice were crossed with miR-203 knock-in mice, where miR-203 expression is induced upon DOX treatment. Thus, organoids derived from the mammary gland tumors of such mouse model where evaluated in vitro. miR-203 expression was therefore induced by Doxycycline in vitro, and compared to other well-defined differentiation media for breast cancer tissue, such as the mammary epithelial cell culture media and kit (CC-2551B; Lonza). Overall design: The general idea was to analyze the transcriptomic proximity between miR-203 transiently-overexpressing organoids and those treated with well-defined differentiation media for breast cancer cells. In these RNA sequencing, the transcriptomic profiles of different clones were tested. Three independent pools of organoids from three different experiments were represented for each sample. Different tumors from the PyMT-miR203 mouse model were tested, named as 474, 476, 496, 366, indicated in the samples description. The treatments tested in vitro for the organoids culture were the following: control; MEGM media (as described above, CC-2551B, Lonza); DOX (to induce miR-203 transient expression in the cells); combinations of the previous treatments. All the treatments were maintained in culture for one week, followed by treatment withdrawal. Organoids were processed for RNA extraction after 3 weeks in culture.

本实验探究了微小RNA-203（microRNA-203）作为潜在分化诱导剂，在源自PyMT小鼠模型的乳腺癌类器官中的调控效果。MMTV-PyVT转基因小鼠在小鼠乳腺肿瘤病毒启动子/增强子的调控下，表达多瘤病毒中间T抗原（Polyoma Virus middle T antigen）。半合子MMTV-PyMT雌性小鼠可形成可触及的乳腺肿瘤，并发生肺转移；与其他乳腺肿瘤模型相比，该品系小鼠的乳腺癌早发外显率更高。

我们将PyMT小鼠与miR-203敲入（knock-in）小鼠杂交，后者的miR-203表达可在多西环素（DOX，Doxycycline）处理后被诱导。因此，我们对源自该杂交小鼠模型乳腺肿瘤的类器官进行了体外评估。

我们通过体外多西环素诱导miR-203表达，并将其与其他用于乳腺癌组织的明确分化培养基进行对比，例如乳腺上皮细胞培养基及配套试剂盒（CC-2551B；Lonza）。

实验整体设计：
本研究的核心思路为分析miR-203瞬时过表达类器官与经明确分化培养基处理的乳腺癌细胞类器官之间的转录组相似性。本次RNA测序（RNA sequencing）实验中，我们对不同克隆的转录组谱进行了检测。每个样本均对应来自3次独立实验的3个独立类器官混合样本。本实验对PyMT-miR203小鼠模型来源的不同肿瘤样本进行了检测，样本编号依次为474、476、496、366，具体信息详见样本说明。

本次体外类器官培养实验中测试的处理方式如下：空白对照组；MEGM培养基（详见上文，CC-2551B；Lonza）；多西环素（DOX，用于诱导细胞中miR-203的瞬时表达）；以及上述处理方式的联合组。所有处理组均在培养体系中维持1周，随后撤除处理因子。培养3周后，收集类器官进行RNA提取。