• Sun. Dec 5th, 2021

** 0.01, 2-way ANOVA. lymphocytes due to GM-CSFCmediated activation of CD103+ dendritic cells and displayed decreased tumor growth and metastasis. GM-CSF neutralization restored tumor growth and metastasis, as did T cell depletion. Importantly, analyses of human tumor data sets support our animal studies. Collectively, these findings demonstrate that endothelial mTORC1 is an actionable target for tumor vessel normalization, which could be leveraged to enhance antitumor immune therapies. = 14C16 mice per group. values were determined by Students tests comparing vehicle- and RAD001-treated groups at day 18. (C and D) Flow cytometric analysis showing low-dose RAD001 treatment decreased p-S6 level in CD45CCD31+ tumor-associated ECs (C) but not in LLC tumor cells (CD45CCD31C) and immune cells (CD45+) (D). Armillarisin A MFI, mean fluorescence intensity. All data are presented as mean SD, and values were determined by 1-way ANOVA with post hoc Tukeys correction for multiple comparisons. ** 0.01, * 0.05. Loss of Raptor/mTORC1 in ECs reduces tumor growth and metastasis. To investigate the role of mTORC1 in vascular ECs genetically, we crossed mice harboring floxed alleles (Raptorfl/fl, referred to as RaptorWT) with mice expressing tamoxifen-inducible Cre recombinase (CreER) under the control of the = 12 to 15 mice per group. ** 0.01, 2-way ANOVA. (D) Representative images of the lungs harvested from WT and RaptorECKO mice after 20 days of LLC tumor implantation. Arrows indicate metastatic foci on the surface of lungs, which were quantified. Armillarisin A (E) Disease-free survival of spontaneous MMTV-PyMT tumors against age (weeks). = 22 to 28 mice per group. ** 0.01. Statistical analysis was performed using log-rank test. (F) Growth curves of spontaneous MMTV-PyMT tumors on WT control and RaptorECKO mice. ** 0.01, 2-way ANOVA. (G) Representative H&E staining of lungs harvested from WT and RaptorECKO/mice. Arrows indicate metastatic foci within the lungs, which were quantified. Scale bar: 200 m. Unless indicated, all data are presented as mean SD, and values Armillarisin A were determined by 2-tailed unpaired Students 2-tailed test. ** 0.01. To complement tumor allograft studies, we analyzed the EC-specific Raptor/mTORC1 loss in the transgenic spontaneous mammary tumor model (33), using RaptorECKO mice crossed with mice (RaptorECKO PyMT). At 8 weeks of age, female RaptorWT PyMT and RaptorECKO PyMT mice were treated with tamoxifen to induce irreversible loss from vascular ECs. Tumor burden was monitored weekly beginning at 18 weeks of age. Notably, mammary tumor latency was delayed (Figure 2E), while tumor growth was markedly reduced (Figure 2F) in tamoxifen-treated RaptorECKO PyMT mice as compared with tamoxifen-treated controls. Further, lung metastasis was significantly inhibited in 28-week-old tamoxifen-treated RaptorECKO PyMT mice as compared with age-matched controls (Figure 2G). These data confirm findings using the LLC allografted tumor model and suggest that Raptor/mTORC1 loss from tumor blood vessels inhibits tumor growth and lung metastasis. Selective inhibition of mTORC1 in ECs decreases angiogenic sprouts and normalizes tumor blood vessels. CR2 To determine the impact of Raptor/mTORC1 on tumor vasculature, we first assessed tumor microvessel density and morphology in situ using CD31 and smooth muscle actin (-SMA), a pericyte marker, to visualize ECs in low-dose RAD001Ctreated LLC-HRE-mCherry-OVA tumors (Figure 3A). Treatment with low-dose RAD001 (0.01 mg/kg) reduced the density of CD31+ tumor vessels (Figure 3B) and induced an increase in pericyte coverage of tumor vessels, as measured by CD31/-SMA costaining in tumors (Figure 3C), indicating an improvement in vessel maturation. Further, measurements of tumor hypoxia using the HRE-mCherry reporter (34) revealed that mCherry expression (Figure 3, D and E) was decreased in LLC-HRE-mCherry-OVA tumors after low-dose RAD001 treatment, and reduced hypoxia was confirmed by the staining of Armillarisin A a hypoxic marker, EF5, on tumor cells (Figure 3F). Taken together, these data suggest that low-dose RAD001 preferentially inhibits mTORC1 signaling in ECs, leading to an increase in tumor vessel normalization. Open in a separate window Figure 3 Selective inhibition of mTORC1 Armillarisin A in endothelium normalizes tumor blood vessels.(A) Representative images of CD31+ (shown in green, EC marker) and -SMA (shown in magenta, pericyte marker) costaining in LLC-HRE-mCherry-OVA tumors treated with low-dose RAD001. Arrows indicate colocalization of CD31+ and -SMA. Scale bar: 100 m. (B) Tumor vessel density was quantified as CD31+ area/field in LLC-HRE-mCherry-OVA tumors. (C) Pericyte coverage on tumor blood vessels was quantified and presented as percentage of -SMA+CD31+ vessels. (D) Representative images of mCherry expression (red) in LLC-HRE-mCherry-OVA tumors treated with low-dose RAD001. Tumor vessels.