Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Aug 6;5(15):e139237.
doi: 10.1172/jci.insight.139237.

Selective inhibition of mTORC1 in tumor vessels increases antitumor immunity

Shan Wang  1   2 , Ariel Raybuck  3 , Eileen Shiuan  4 , Sung Hoon Cho  3 , Qingfei Wang  5 , Dana M Brantley-Sieders  2   6 , Deanna Edwards  2 , Margaret M Allaman  7 , James Nathan  8 , Keith T Wilson  1   3   4   6   7 , David DeNardo  9 , Siyuan Zhang  5 , Rebecca Cook  3   7   10 , Mark Boothby  3   4   6 , Jin Chen  1   2   4   6   10
Affiliations

Selective inhibition of mTORC1 in tumor vessels increases antitumor immunity

Shan Wang et al. JCI Insight. .

Abstract

A tumor blood vessel is a key regulator of tissue perfusion, immune cell trafficking, cancer metastasis, and therapeutic responsiveness. mTORC1 is a signaling node downstream of multiple angiogenic factors in the endothelium. However, mTORC1 inhibitors have limited efficacy in most solid tumors, in part due to inhibition of immune function at high doses used in oncology patients and compensatory PI3K signaling triggered by mTORC1 inhibition in tumor cells. Here we show that low-dose RAD001/everolimus, an mTORC1 inhibitor, selectively targets mTORC1 signaling in endothelial cells (ECs) without affecting tumor cells or immune cells, resulting in tumor vessel normalization and increased antitumor immunity. Notably, this phenotype was recapitulated upon targeted inducible gene ablation of the mTORC1 component Raptor in tumor ECs (RaptorECKO). Tumors grown in RaptorECKO mice displayed a robust increase in tumor-infiltrating lymphocytes due to GM-CSF-mediated 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.

Keywords: Cancer immunotherapy; Immunology; Oncology; endothelial cells.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Low-dose RAD001 selectively inhibits mTORC1 signaling in tumor endothelium and suppresses tumor growth.
(A) Schematic diagram showing the experimental design with LLC-HRE-mCherry-OVA tumor cell implantation, RAD001 treatment, adoptive T cell transfer, and EF5 intravenous injection. (B) Growth curves of LLC-HRE-mCherry-OVA tumors treated with a low dose of RAD001. n = 14–16 mice per group. P values were determined by Student’s t 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 CD45–CD31+ tumor-associated ECs (C) but not in LLC tumor cells (CD45–CD31–) and immune cells (CD45+) (D). MFI, mean fluorescence intensity. All data are presented as mean ± SD, and P values were determined by 1-way ANOVA with post hoc Tukey’s correction for multiple comparisons. **P ≤ 0.01, *P ≤ 0.05.
Figure 2
Figure 2. Raptor/mTORC1 loss in tumor endothelium decreases tumor growth and metastasis.
(A) Schematic diagram showing the experimental procedure of tamoxifen treatment and subcutaneous implantation of LLC tumor nodules. (B) Representative image of bioluminescence signal from LLC tumors on WT control and RaptorECKO mice. (C) Growth curves of LLC tumors on WT control and RaptorECKO mice. Tumors were measured by a caliper every other day from day 10 through 20 after tumor implantation. n = 12 to 15 mice per group. **P ≤ 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. (E) Disease-free survival of spontaneous MMTV-PyMT tumors against age (weeks). n = 22 to 28 mice per group. **P ≤ 0.01. Statistical analysis was performed using log-rank test. (F) Growth curves of spontaneous MMTV-PyMT tumors on WT control and RaptorECKO mice. **P ≤ 0.01, 2-way ANOVA. (G) Representative H&E staining of lungs harvested from WT and RaptorECKO/MMTV-PyMT mice. Arrows indicate metastatic foci within the lungs, which were quantified. Scale bar: 200 μm. Unless indicated, all data are presented as mean ± SD, and P values were determined by 2-tailed unpaired Student’s 2-tailed t test. **P ≤ 0.01.
Figure 3
Figure 3. Selective inhibition of mTORC1 in endothelium normalizes tumor blood vessels.
(A) Representative images of CD31+ (shown in green, EC marker) and α-SMA (shown in magenta, pericyte marker) costaining in LLC-HRE-mCherry-OVA tumors treated with low-dose RAD001. Arrows indicate colocalization of CD31+ and α-SMA. Scale bar: 100 μm. (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 α-SMA+CD31+ vessels. (D) Representative images of mCherry expression (red) in LLC-HRE-mCherry-OVA tumors treated with low-dose RAD001. Tumor vessels were assessed by CD31 staining (green). Arrows indicate mCherry+ hypoxic area. Scale bar: 50 μm. (E and F) Hypoxic regions in LLC-HRE-mCherry-OVA tumors were quantified by flow cytometry to assess the fluorescence intensity of mCherry+ (E) and EF5+ (F) in CD45– tumor cells after RAD001 treatment. (G) Representative images and quantification of CD31+ blood vessels (red) in LLC tumors harvested from WT control and RaptorECKO mice. n = 5–7 mice per group. Scale bar: 100 μm. (H) Representative images and quantification of lumen size of CD31+ vessels from WT and RaptorECKO tumors. Zoomed-in images (original magnification, ×20) of dotted-line area are shown at the bottom. White solid lines mark lumen area in CD31+ vessels. n = 3 mice per group. Scale bar: 100 μm. (I) Costain of CD31+ (red) and α-SMA (green) in LLC tumors from WT control and RaptorECKO mice. Arrows indicate colocalization of CD31 and SMA. Pericyte coverage on tumor blood vessels was quantified and presented as percentage of α-SMA+CD31+ vessels. n = 3–4 mice per group. Scale bar: 100 μm. (J) Representative images showing lectin perfusion (green) in CD31+ tumor blood vessels (red). Arrows indicate lectin-perfused functional blood vessels. Vessel perfusion was quantified and presented as percentage of Lectin+CD31+/total CD31+ vessels. n = 5 mice per group. Scale bar: 100 μm. (K) Hypoxia was assessed by mCherry expression (red) in WT control and RaptorECKO tumors and quantified as mCherry+ intensity within tumors. n = 5–6 mice per group. Scale bar: 50 μm. AU, arbitrary units. All data are presented as mean ± SD. **P ≤ 0.01. *P ≤ 0.05, Student’s 2-tailed t test.
Figure 4
Figure 4. Low-dose RAD001 increases the numbers and effector function of infiltrating T lymphocytes.
(A) Schematic diagram showing the experimental design with LLC-HRE-mCherry-OVA tumor cell implantation, RAD001 treatment, and adoptive T cell transfer. (B and C) Representative flow cytometric plots and quantification of tumor-infiltrating CD45+ immune cells (B) and TCRβ+ T cells (C) in LLC tumors treated with low-dose RAD001. (D and E) Representative flow cytometric plots and quantification of Thy1.1+CD8+ (D) and CD8+YFP+ (IFN-γ+) (E) donor T cells in LLC tumors treated with low-dose RAD001. (F and G) Representative flow cytometric plots and quantification of Thy1.1+CD4+ (F) and CD4+YFP+ (IFN-γ+) (G) donor T cells in LLC tumors treated with low-dose RAD001. All data are presented as mean ± SD. **P ≤ 0.01. *P ≤ 0.05, Student’s 2-tailed t test.
Figure 5
Figure 5. Raptor/mTORC1 loss in endothelium increases the numbers and effector function of tumor-infiltrating T cells.
(A) Schematic diagram of experimental design with LLC tumor allograft. (B) Tumor weight at day 18 after implantation. Each dot represents a mouse. (C and D) Flow cytometric analysis of CD45+ (C) and IFN-γ+CD8+ (D) immune cells in WT and RaptorECKO tumors. (E) Schematic diagram of experimental design in MMTV-PyMT-OVA tumor orthotopic allograft. (F) Tumor weight at day 14 after implantation. (G) Flow cytometric analysis of donor (CD45.1) and recipient (CD45.2) CD45+ immune cells in WT and RaptorECKO tumors. Donor CD45.1+ cells were quantified and shown on the right. (H) Immunofluorescence images of CD45.1 (green) CD8 OT-I donor T cells costained with CD31 (red) in PyMT tumors. Numbers of CD45.1+ cells were quantified. Arrows indicate CD45.1+ donor T cells in the LLC tumor. Scale bar: 100 μm. (I and J) Flow cytometric analysis of IFN-γ (I) and GZMB (J) in CD8+ T cells in WT and RaptorECKO PyMT-OVA tumors. (K) Schematic diagram of experimental design in LLC tumors treated with anti-CD4 and anti-CD8 neutralizing antibodies. (L) Growth curves of LLC tumors on WT control and RaptorECKO mice after T cell depletion. n = 3–7 mice per group. Two-way ANOVA. (M) T cell depletion was confirmed by flow cytometric analysis. Unless indicated, all data are presented as mean ± SD. **P ≤ 0.01. *P ≤ 0.05, Student’s 2-tailed t test.
Figure 6
Figure 6. GM-CSF is required for increase in CD103+ DCs and IFN-γ+CD8+ T cells in RaptorECKO tumors.
(A) Heatmap showing relative expression levels of indicated chemokines/cytokines in LLC tumors harvested from 6 sex-matched littermate pairs of WT and RaptorECKO mice. Red indicates higher expression while green indicates lower expression in RaptorECKO tumors over WT control. (B) Quantification of GM-CSF expression in RaptorECKO tumors and WT control tumors from Luminex analysis. (C) GM-CSF ELISA on independent LLC tumor lysates to verify elevated GM-CSF expression in RaptorECKO tumors. n = 8 mice per group. (D–F) Flow cytometric analysis of CD11b+, CD11b+CD11c+, or CD11c+ immune cells and CD103+ DCs in WT and RaptorECKO tumors from the LLC model (D) and PyMT model (E) or in LLC-HRE-mCherry-OVA tumors treated with low-dose RAD001 (F). (G) Schematic diagram showing the experimental procedure with GM-CSF neutralizing antibody treatment in the LLC model. (H) Representative image of bioluminescence signal from LLC tumors on RaptorECKO mice treated with anti–GM-CSF or IgG control on day 20 postimplantation. (I) Growth curves of LLC tumors on WT mice treated with IgG and RaptorECKO mice treated with anti–GM-CSF or IgG isotype antibodies. n = 8–11 mice per group. **P ≤ 0.01. Two-way ANOVA. (J) Quantification of metastatic foci on the surface of lungs harvested in I. (K and L) Flow cytometric analysis of CD11b+, CD11b+CD11c+, or CD11c+ immune cells and CD103+CD11c+ DCs (K) and IFN-γ+CD8+ T cells (L) in tumors treated with IgG control and tumors treated with anti–GM-CSF. Unless indicated, all data are presented as mean ± SD. **P ≤ 0.01. *P ≤ 0.05, Student’s 2-tailed t test.
Figure 7
Figure 7. Correlations between vessel normalization markers and mTORC1-mediated signaling, as well as GM-CSF and immune markers in human tumor samples.
(A and B) Correlation between vessel normalization markers and mTORC1-mediated signaling using non–small cell lung cancer (NSCLC, GSE127465) (A) or triple-negative breast cancer (TNBC, GSE118390) (B) scRNA-Seq data sets. The x and y axes represent ssGSEA enrichment scores of indicated gene sets in ECs. Each point represents a single EC. (C and D) Correlations between CSF-2 (GM-CSF) transcript levels and levels of an immune cell marker PTPRC (CD45) (C) and a cytotoxic activity signature (an average expression of IFNG, GZMB, and PRF1) (D) in lung tumors (top) and breast tumors (bottom). (E) Correlations between CSF-2 (GM-CSF) transcript levels and levels of BATF3, IRF8, and CCR7 in lung tumors (top panels) and breast tumors (bottom panels). BATF3, IRF8, and CCR7 are “signature genes” for the human CD103+ DC population. The x and y axes are log2(mRNA) expression using the Lung Cancer (LUNG) data set (n = 1129) and the Breast Cancer (BRCA) cohort (n = 1218) from TCGA. Each dot represents a single tumor sample. Pearson correlation coefficient (r) and 2-tailed P values are shown.
See this image and copyright information in PMC

References

    1. Huang Y, Kim BYS, Chan CK, Hahn SM, Weissman IL, Jiang W. Improving immune-vascular crosstalk for cancer immunotherapy. Nat Rev Immunol. 2018;18(3):195–203. doi: 10.1038/nri.2017.145. - DOI - PMC - PubMed
    1. Rohlenova K, Veys K, Miranda-Santos I, De Bock K, Carmeliet P. Endothelial cell metabolism in health and disease. Trends Cell Biol. 2018;28(3):224–236. doi: 10.1016/j.tcb.2017.10.010. - DOI - PubMed
    1. Dewhirst MW, Secomb TW. Transport of drugs from blood vessels to tumour tissue. Nat Rev Cancer. 2017;17(12):738–750. doi: 10.1038/nrc.2017.93. - DOI - PMC - PubMed
    1. Schaaf MB, Garg AD, Agostinis P. Defining the role of the tumor vasculature in antitumor immunity and immunotherapy. Cell Death Dis. 2018;9(2):115. doi: 10.1038/s41419-017-0061-0. - DOI - PMC - PubMed
    1. Hendry SA, Farnsworth RH, Solomon B, Achen MG, Stacker SA, Fox SB. The role of the tumor vasculature in the host immune response: implications for therapeutic strategies targeting the tumor microenvironment. Front Immunol. 2016;7:621. - PMC - PubMed

Publication types

MeSH terms

Substances