Abstract 2393: Characterization and validation of a Smad3/TGFb pathway reporter mouse for analysis of TGFβ signaling in normal homeostasis and cancer
Authors: Yang Yuan , Millman Zachary , Stuelten Christina , Gargesha Madhu , Simpson Mark , Yang Howard , Lee Maxwell , Wakefield Lalage M. 
Published: April 2023

Abstract
TGFβs play a central regulatory role in maintaining homeostasis in the adult animal, and dysregulation of TGFβ signaling occurs in many cancers. Consequently, TGFβ pathway antagonists are now in early phase clinical oncology trials, but surprisingly little is known about when and where the TGFβ pathway is activated in the adult animal. We recently generated a TGFβ pathway reporter mouse in which expression of eGFP is driven by an enhancer consisting of 6 repeats of a strong Smad3 binding element (S3x6&gt;GFP reporter), knocked into the ROSA26 locus. Here we present further characterization of the founder line (“Lime” mouse). Whole body fluorescent imaging of an adult mouse highlighted Smad3 activation in the expected tissues, such as the gastrointestinal tract, costal cartilage, and brown adipose tissue among others. Unexpectedly, intercrossing the Lime mouse with a Smad3 germline knockout mouse did not reveal a significant reduction in signal. However, since Smad3 knockout mice survive to adulthood despite having no Smad3, the persistence of signal could reflect compensatory signaling through other Smads, such as Smad1/5 or the Smad3-like Smad2 splice variant, Smad2DelEx3. Alternatively, the reporter may not report with fidelity in the chromatin context of the ROSA26 locus. To address these alternatives, Lime mice were intercrossed with the MMTV-PyVT model of mammary tumorigenesis. Mammary tumors showed high reporter activity, and a mammary tumor cell line (“LimePyVT”) was derived for characterization. Reporter activity in LimePyVT cells was enhanced by TGFβ and reduced by TGFβ receptor kinase inhibitors, but not by a BMP kinase inhibitor, as expected. Reduction in reporter signal with TGFβ receptor kinase inhibitors was only seen with prolonged treatment and 2x daily redosing, likely reflecting both the long half-life of the GFP (1-2 days), and the high sensitivity of the reporter to breakthrough signaling. siRNA knockdown in LimePyVT cells ex vivo confirmed that GFP reporter expression is dependent on Smad3 and Smad4. In other validation experiments, multicolor immunofluorescence showed heterogeneous reporter activity in the normal mammary epithelium, with highest activity in ER+ cells, where TGFβ is most active. Normal mammary epithelial cells were FACS-sorted into GFP-high and GFP-low fractions. RNASeq and pathway analysis showed TGFβ to be the top upstream regulator of the GFP-high cell transcriptome, consistent with the reporter reflecting TGFβ pathway activation. Overall, we believe that the high sensitivity of the reporter and the long GFP half-life likely mean that it will not report accurately on the impact of TGFβ antagonists in vivo. However, this reporter mouse should be a useful tool to assess the cellular location and extent of TGFβ/activin pathway activation during tumor progression, and the transcriptomic consequences.
Citation Format: Yuan Yang, Zachary Millman, Christina Stuelten, Madhu Gargesha, Mark Simpson, Howard Yang, Maxwell Lee, Lalage M. Wakefield. Characterization and validation of a Smad3/TGFb pathway reporter mouse for analysis of TGFβ signaling in normal homeostasis and cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2393.