American Association for Cancer Research
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Supplementary Video 10 from Basal but not Luminal Mammary Epithelial Cells Require PI3K/mTOR Signaling for Ras-Driven Overgrowth

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posted on 2023-03-30, 21:24 authored by Kristin A. Plichta, Jessica L. Mathers, Shelley A. Gestl, Adam B. Glick, Edward J. Gunther

MOV file - 2.4MB, Effects of mTor Inhibitor rapamycin on Ras-driven luminal MEC overgrowth. Representative video depicting an organoid prepared from a Dox-naive luminalRas/GFP (MMTV-rtTA/TGFP/TRAS) mouse cultured in the presence of both Dox and rapamycin in 3D



The mammary ducts of humans and mice are comprised of two main mammary epithelial cell (MEC) subtypes: a surrounding layer of basal MECs and an inner layer of luminal MECs. Breast cancer subtypes show divergent clinical behavior that may reflect properties inherent in their MEC compartment of origin. How the response to a cancer-initiating genetic event is shaped by MEC subtype remains largely unexplored. Using the mouse mammary gland, we designed organotypic three-dimensional culture models that permit challenge of discrete MEC compartments with the same oncogenic insult. Mammary organoids were prepared from mice engineered for compartment-restricted coexpression of oncogenic H-RASG12V together with a nuclear fluorescent reporter. Monitoring of H-RASG12V-expressing MECs during extended live cell imaging permitted visualization of Ras-driven phenotypes via video microscopy. Challenging either basal or luminal MECs with H-RASG12V drove MEC proliferation and survival, culminating in aberrant organoid overgrowth. In each compartment, Ras activation triggered modes of collective MEC migration and invasion that contrasted with physiologic modes used during growth factor–initiated branching morphogenesis. Although basal and luminal Ras activation produced similar overgrowth phenotypes, inhibitor studies revealed divergent use of Ras effector pathways. Blocking either the phosphoinositide 3-kinase or the mammalian target of rapamycin pathway completely suppressed Ras-driven invasion and overgrowth of basal MECs, but only modestly attenuated Ras-driven phenotypes in luminal MECs. We show that MEC subtype defines signaling pathway dependencies downstream of Ras. Thus, cells-of-origin may critically determine the drug sensitivity profiles of mammary neoplasia. Cancer Res; 72(22); 5856–66. ©2012 AACR.