Basal but not Luminal Mammary Epithelial Cells Require PI3K/mTOR Signaling for Ras-Driven Overgrowth

H-RAS^G12V expressed in either MEC compartment drives organoid overgrowth. A, normal histomorphology of Dox-naïve transgenic mammary glands. Representative images of camine-stained mammary gland whole mounts and immunohistochemically stained tissue sections are shown. White bar, 500 μm; black bar, 100 μm. B, time-lapse imaging of organoid overgrowth. Organoids prepared from Dox-naïve mice engineered for Ras pathway activation in either basal MECs (K5-rtTA/TGFP/TRAS; Basal^RAS/GFP) or luminal MECs (MMTV-rtTA/TGFP/TRAS; Luminal^RAS/GFP) were cultured concurrently in 3D along with genetic control organoids lacking the TRAS transgene (Basal^GFP and Luminal^GFP). Sequential images of representative organoids of each genotype captured during a 1 week live-cell imaging experiment are shown. Times denote hrs after Dox addition. Bar, 50 μm. C, Ras-mediated changes in organoid size and shape. 2D organoid images captured after 6 days of Dox treatment were subjected to morphometric analysis to determine mean organoid area (left) and circularity (right). Mean values reflect analysis of 23 to 111 organoids for each genotype and treatment condition. D, Ras-mediated increases in MEC proliferation and survival. Organoid videos were viewed frame-by-frame to score mitotic (left) and cell death (right) events occurring during the first 2 days of Dox treatment. Mean values reflect indices (events per organoid, normalized to a 24-hour acquisition period) derived from analyzing videos from 8 to 38 organoids of each genotype. *, P < 0.01; **, P < 0.05; Kruskal–Wallis analysis of variance, Tukey–Kramer method. All error bars indicate SEM.

H-RAS^G12V, but not Fgf2, triggers focal invasion into Matrigel. A, Fgf2-induced multilayered epithelial elongation. Still image from a live cell imaging experiment depict a representative luminal^GFP organoid 7 days after initiating Fgf2-induced branching morphogenesis. B, Ras-mediated focal invasion. Time-lapse images depict leader cells initiating invasion. Arrowheads mark protrusions emanating from leader cells indicated by white arrows. Red arrows indicate follower “stalk” cells. Bar, 25μm. C, quantification of Ras-mediated focal invasion. Organoids of the indicated genotypes were cultured in the presence and absence of Dox for 6 days, then scored for the presence of focal invasion as described in Materials and Methods. Mean values reflect analysis of 20 to 77 organoids for each genotype and treatment condition. D, contrasting modes of collective MEC invasion triggered by Fgf2 versus H-RAS^G12V. Organoids of the indicated genotypes underwent Fgf2-induced branching before Dox treatment. Panels depict morphology of a representative organoid before and after Dox-induced transgene expression.

MEK inhibition partially blocks Ras phenotypes in both MEC compartments. Basal^RAS/GFP and luminal^RAS/GFP organoids were cultured under the indicated treatment conditions for 6 days. PD denotes MEK inhibitor PD0325901 at 100 nmol/L. Left panels depict day 6 images (×10) of representative organoids for each genotype and treatment condition (see also Videos 5 and 6). Morphometry of 2D images captured at days 0, 2, 4, and 6 were used to determine the prevalence of focal invasion as well as the mean organoid circularity and area at each time point. Each data point reflects the analysis of 37 to 55 organoids. Error bars denote SEM.

PI3K/mTOR inhibition fully blocks Ras phenotypes in basal but not luminal MECs. Basal^RAS/GFP and luminal^RAS/GFP organoids were cultured under the indicated treatment conditions for 6 days. Left panels depict day 6 images (×10) of representative organoids for each genotype and treatment condition (see also Videos 7–10). Morphometry of 2D images captured at days 0, 2, 4, and 6 were used to determine the prevalence of focal invasion as well as the mean organoid circularity and area at each time point. A, PI3K pathway inhibition. LY denotes PI3K inhibitor LY294002 at 10 μmol/L. Each data point reflects the analysis of 24 to 47 organoids. B, mTOR pathway inhibition. Rap denotes Rap at 5 nmol/L. Each data point reflects the analysis of 18 to 114 organoids. Error bars denote SEM.

Suppressing Ras-driven phenotypes involves decreased proliferation and preservation of MEC compartments. Basal^RAS/GFP and luminal^RAS/GFP organoids were cultured under the indicated treatment conditions in 3D. Mitotic and cell death events were scored during the first 2 days of Dox treatment. A, MEK inhibitor effects on MEC proliferation and survival. PD denotes MEK inhibitor PD0325901 at 100 nmol/L. B, PI3K/mTOR inhibitor effects on MEC proliferation and survival. Mean values reflect indices (events per organoid, normalized to a 24-hour acquisition period) derived from analyzing videos from 11 to 34 organoids of each genotype. Error bars indicate SEM. C, PI3K/mTOR inhibitor effects on MEC compartmentalization. Organoids were recovered after 3D culture for 7 days, then fixed and stained with Hoechst and phalloidin. Panels depict equatorial confocal microscopy images (×63) of representative organoids of each indicated genotype and treatment condition. Bars, 50 μm. LY denotes PI3K inhibitor LY294002 at 10 μmol/L. Rap denotes Rap at 5 nmol/L.