Morphological screening of mesenchymal mammary tumor organoids to identify drugs that reverse epithelial-mesenchymal transition

Na Zhao,Sendurai A Mani,Clifford C Stephan,Goeun Bae,Hannah L Johnson,Martina Strempfl,Kevin P Roarty,Michael J Toneff,Fabio Stossi,Reid T Powell,Jeffrey M Rosen,Xueying Yuan,Philip Jones

doi:10.1038/s41467-021-24545-3

Abstract

The epithelial-mesenchymal transition (EMT) has been implicated in conferring stem cell properties and therapeutic resistance to cancer cells. Therefore, identification of drugs that can reprogram EMT may provide new therapeutic strategies. Here, we report that cells derived from claudin-low mammary tumors, a mesenchymal subtype of triple-negative breast cancer, exhibit a distinctive organoid structure with extended “spikes” in 3D matrices. Upon a miR-200 induced mesenchymal-epithelial transition (MET), the organoids switch to a smoother round morphology. Based on these observations, we developed a morphological screening method with accompanying analytical pipelines that leverage deep neural networks and nearest neighborhood classification to screen for EMT-reversing drugs. Through screening of a targeted epigenetic drug library, we identified multiple class I HDAC inhibitors and Bromodomain inhibitors that reverse EMT. These data support the use of morphological screening of mesenchymal mammary tumor organoids as a platform to identify drugs that reverse EMT.

Highlights

The epithelial-mesenchymal transition (EMT) has been implicated in conferring stem cell properties and therapeutic resistance to cancer cells
We first established several primary cell lines from Trp53-null murine mammary tumor models. These Trp53-null genetically modified mouse (GEM) tumors were previously established by transplantation of donor mammary epithelium from BALB/c mice, where Trp[53] was deleted from the germline, into syngeneic hosts for derivation of a variety of Trp53-null mammary tumors[41,42]
The inducible expression of the mesenchymal-epithelial transition (MET) promoter miR-200c by addition of doxycycline to TetOn-miR-200c cells prevented the formation of these invasive protrusions without obviously affecting organoid size (Fig. 1b, Supplementary Fig. 1a, b, and Supplementary Movie 1), and markedly induced the expression of luminal cytokeratin K8 at both RNA and protein levels (Fig. 1c and Supplementary Fig. 1c–e)

Summary

Introduction

The epithelial-mesenchymal transition (EMT) has been implicated in conferring stem cell properties and therapeutic resistance to cancer cells. In a preclinical claudin-low/mesenchymal genetically modified mouse (GEM) model developed in our laboratory, re-expression of the miR-200 microRNAs, which are master regulators of the mesenchymal-epithelial transition (MET)[10,11,12], reversed cancer stem cell properties and sensitized tumors to chemotherapy[13]. We developed a screening approach that combines generic image features from a pre-trained dNN with a k-nearest neighbors (k-NN) model to map the similarity of experimental treatments to reference controls Using this approach, we identified a number of small molecule inhibitors that reversed EMT in an epigenetic drug screen. Such screens serve as a first step to provide insights into the epigenetic regulation of the EMT phenotype

Methods

Results

Conclusion