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). 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.