c-Met-mediated endothelial plasticity drives aberrant vascularization and chemoresistance in glioblastoma - PubMed (original) (raw)

. 2016 May 2;126(5):1801-14.

doi: 10.1172/JCI84876. Epub 2016 Apr 4.

Tianrun Liu, Peihong Ma, R Alan Mitteer Jr, Zhenting Zhang, Hyun Jun Kim, Eujin Yeo, Duo Zhang, Peiqiang Cai, Chunsheng Li, Lin Zhang, Botao Zhao, Laura Roccograndi, Donald M O'Rourke, Nadia Dahmane, Yanqing Gong, Constantinos Koumenis, Yi Fan

c-Met-mediated endothelial plasticity drives aberrant vascularization and chemoresistance in glioblastoma

Menggui Huang et al. J Clin Invest. 2016.

Abstract

Aberrant vascularization is a hallmark of cancer progression and treatment resistance. Here, we have shown that endothelial cell (EC) plasticity drives aberrant vascularization and chemoresistance in glioblastoma multiforme (GBM). By utilizing human patient specimens, as well as allograft and genetic murine GBM models, we revealed that a robust endothelial plasticity in GBM allows acquisition of fibroblast transformation (also known as endothelial mesenchymal transition [Endo-MT]), which is characterized by EC expression of fibroblast markers, and determined that a prominent population of GBM-associated fibroblast-like cells have EC origin. Tumor ECs acquired the mesenchymal gene signature without the loss of EC functions, leading to enhanced cell proliferation and migration, as well as vessel permeability. Furthermore, we identified a c-Met/ETS-1/matrix metalloproteinase-14 (MMP-14) axis that controls VE-cadherin degradation, Endo-MT, and vascular abnormality. Pharmacological c-Met inhibition induced vessel normalization in patient tumor-derived ECs. Finally, EC-specific KO of Met inhibited vascular transformation, normalized blood vessels, and reduced intratumoral hypoxia, culminating in suppressed tumor growth and prolonged survival in GBM-bearing mice after temozolomide treatment. Together, these findings illustrate a mechanism that controls aberrant tumor vascularization and suggest that targeting Endo-MT may offer selective and efficient strategies for antivascular and vessel normalization therapies in GBM, and possibly other malignant tumors.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Mesenchymalization in human GBM-associated ECs.

Single-cell suspensions were prepared from surgical specimens of GBM patient tumors (n = 15 patients). (A and B) ECs were isolated from the cell suspensions by CD31 antibody–based magnetic-activating cell sorting. (A) Cell lysates were resolved by SDS-PAGE, followed by immunoblot analysis. Representative data are shown from 3 independent experiments. (B) Cells were stained with anti–FSP-1 antibody and analyzed by flow cytometry. Representative data are shown from 6 patients. (C) Whole-tumor cell suspensions were stained with anti–FSP-1 and -CD31 or control IgGs and analyzed by flow cytometry. Representative data are shown from 3 patients. (D) Sections of surgical specimens from normal brain and GBM patients were probed with anti–FSP-1, -CD105, and -CD31 antibodies. Left, representative images. Right, quantitative analysis of colocalization of CD31+ ECs with FSP-1+ fibroblast–like cells (n = 5 patients, mean ± SEM, unpaired t test). Scale bar: 50 μm.

Figure 2

Figure 2. Robust Endo-MT in GBM vasculature.

(A and B) GBM was induced by orthotopic injection of retrovirus or GL26 tumor cells. The GBM in Ntv-a Ink4a_–/–_Ptenfl/fl LSL-Luc mice is induced by PDGF overexpression and Cre-mediated Pten deletion in neural stem and progenitor cells through RCAS-mediated somatic gene transfer. (A) Schematic approach. (B) Single-cell suspension from normal brain and tumors was stained with anti-CD31 and anti–FSP-1 antibodies and analyzed by flow cytometry. Left, representative cell sorting. Right, quantitative analysis (mean ± SEM, n = 6–8, unpaired t test). (C–E) The primary GBM in Ntv-a Ink4a_–/–_Ptenfl/fl LSL-Luc donor mice is induced by RCAS-mediated somatic gene transfer. Recipient mice were WT and Tie2-Cre mice. (C) Schematic approach. (D) Single-cell suspensions derived from tumors were stained with anti-Cre and anti–FSP-1 antibodies and analyzed by flow cytometry. Left, representative cell sorting. Right, quantitative analysis (mean ± SEM, n = 6, unpaired t test). (E) Tumor sections were subjected to immunofluorescence analyses. Right, enlarged images of left rectangles. Representative data are shown from 4–5 mice. Scale bars: 100 μm.

Figure 3

Figure 3. Endo-MT induces vascular abnormality in vitro.

(A–F, and H) Human brain microvascular ECs were treated with control ECM or U251 glioma-CM. (A) Twenty-four hours after treatment, cells were stained with anti–VE-cadherin antibody and costained with Alexa Fluoro 567–labeled phalloidin for visualization of F-actin. Representative data are shown from 2 independent experiments. Scale bars: 100 μm. (B) ECs were lysed. Cell lysates were resolved by SDS-PAGE and immunoblotted. Representative data are shown from 3 independent experiments. (C) mRNA was isolated from ECs 24 hours after treatment and analyzed by qPCR (mean ± SEM, n = 3). (D–F) ECs pretreated with control medium or glioma-CM for 24 hours were trypsinized and cultured in normal culture medium. (D) Cell proliferation was determined with MTT assay. Cell migration was determined with transwell assay. Cell invasion across Matrigel-coated membranes in tranwells was determined. Data expressed as the percentage of control (mean ± SEM, n = 5, paired t test). (E) EC monolayer permeability was analyzed by measuring the fluorescence of diffused FITC-dextran across transwell membrane (mean ± SEM, n = 6, paired t test). (F) Tube formation was induced on Matrigel. Left, representative images. Right, quantified total tube length (mean ± SEM, n = 6, paired t test). Scale bar: 200 μm. (G) ECs isolated from normal brain or patient GBM tumor were subjected to cell viability analysis (mean ± SEM, n = 4, unpaired t test). (H) Cells pretreated with control medium or glioma-CM for 48 hours were incubated with Dil-Ac-LDL and stained with anti–FSP-1 antibody. Representative data are shown from 3 independent experiments. Scale bar: 50 μm.

Figure 4

Figure 4. c-Met is a key regulator of Endo-MT.

(A–C) Human brain microvascular ECs were treated with control ECM medium or U251 glioma-CM. (A) Cells were treated for 8 hours, and cell lysates were analyzed with a phospho-receptor tyrosine kinase array. (B) Cells were treated with glioma-CM for different time periods and subjected to immunoblot analysis. (C) Cells were treated with 5 μM c-Met inhibitor SU11274, EGFR inhibitor erlotinib, and TGF-β1 inhibitor SB431542 or 0.1% DMSO (vehicle) in glioma-CM for 24 hours and subjected to immunoblot analysis. (D) Cells were treated with 25 ng/ml HGF, 100 ng/ml EGF, 10 ng/ml VEGF-A, or 10 ng/ml TGF-β1 for 6 days. Cell lysates were analyzed by immunoblot. (E and F) Cells were treated with glioma-CM that was harvested from U251 (E), U87 (F), and primary patient 5377 GBM cells, in the presence of 20 μg/ml control IgG or anti-HGF antibody. Cell lysates were subjected to immunoblot analysis. (G) Cells transduced with control or c-Met shRNA were treated with control ECM medium and U251 glioma-CM for 24 hours. Cell lysates were analyzed by immunoblot. All representative blots are shown from 2–3 independent experiments.

Figure 5

Figure 5. c-Met is required for glioma-CM–induced vascular abnormalities.

(A–D) Human brain microvascular ECs were transduced with lentivirus that encodes control or c-Met shRNA and treated with control ECM medium and U251 glioma-CM for 24 hours. (A–C) After treatment, cells were trypsinized and cultured in normal medium. (A) Cell proliferation was determined by MTT-based assay (mean ± SEM, n = 6, paired t test). (B) Cells were seeded on transwell membranes and subjected to migration analysis (mean ± SEM, n = 6, paired t test). (C) EC monolayer permeability was analyzed by measuring the fluorescence of diffused FITC-dextran across transwell membrane (mean ± SEM, n = 6, paired t test). (D) Tube formation was induced on Matrigel. Representative data are shown from 3 independent experiments. Scale bar: 200 μm. (E) ECs isolated from normal human brain and GBM tumor of patient 5377 were pretreated with 5 μM SU11274 or 0.1% DMSO (vehicle). Tube formation was induced on Matrigel. Representative data are shown from 4 patients. Scale bar: 200 μm.

Figure 6

Figure 6. c-Met activation induces ETS-dependent MMP-14 expression, leading to VE-cadherin cleavage and Endo-MT.

(A) Human brain microvascular ECs were treated with U251 glioma-CM and analyzed by immunoblot. (B) Cells were lentivirally transduced with c-Met or control shRNA and treated with glioma-CM. Cell lysates were subjected to immunoblot analysis. (C and D) Glioma-CM–induced FSP-1 or α-SMA expression is inhibited by knockdown of MMP-14 but not MMP-2. Cells transduced with MMP-14 (C), MMP-2 (D), or control shRNA were treated with glioma-CM and analyzed by immunoblot. (E) Cells were transfected with NF-κB, ETS-1, or control siRNA and treated with 100 ng/ml HGF. Cell lysates were subjected to immunoblot analysis. (F and G) Cells were incubated with active MMP-14 for 18 hours. (F) Culture medium and cell lysates were immunoblotted with anti–VE-cadherin antibody. (G) MMP-14 induces VE-cadherin disorganization and downregulation. Cells were subjected to immunofluorescence analysis with an anti–VE-cadherin antibody. Scale bar: 20 μm. (H) Cells were lentivirally transduced with c-Met or control shRNA, treated with glioma-CM in the presence or absence of active MMP-14, and analyzed by immunoblot. (I) ECs isolated from normal brain and patient GBM tumors were treated with 5 μM SU11274 or 0.1% DMSO (vehicle) and subjected to immunoblot analysis. All representative data are shown from 2–3 independent experiments.

Figure 7

Figure 7. c-Met is critical for cancer progression and chemoresistance in GBM.

(A) Angiogenesis analysis using Metfl/fl mice with or without expression of Tie2–Cre. Mouse embryos (left) at E14.5 were imaged, and brain tissues (right) were probed with anti-vWF antibody. Representative data are shown from 6 mouse embryos. Scale bar: 100 μm. (B) Aortic ECs were isolated from Metfl/fl and Tie2-Cre Metfl/fl mice and treated with GL26 glioma-CM. Cell lysates were immunoblotted. Representative data are shown from 2 independent experiments. (C–F) The genetic GBM model was induced. Metfl/fl and Tie2-Cre Metfl/fl mice were injected with primary GBM cells and treated with saline or 100 mg/kg TMZ 14 days after tumor transplantation. (C) Schematic model of experimental approaches. (D) Met deletion in ECs increases mouse survival with TMZ chemotherapy. Survival was monitored for 60 days after injection (total n = 57 mice, pooled from 2 independent experiments). MST, median survival time. (E) Met deletion in ECs inhibits tumor growth with TMZ chemotherapy. Tumor growth was analyzed by whole-body bioluminescence imaging. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 20 (n = 8–13, mean ± SEM, unpaired t test). (F) Tumor sections were stained with H&E and imaged. Representative data are shown from 8 mice/group. Scale bar: 100 μm.

Figure 8

Figure 8. c-Met is critical for Endo-MT and aberrant vascularization in GBM.

The genetic GBM model was induced in Metfl/fl and Tie2-Cre Metfl/fl mice and treated with saline or 100 mg/kg TMZ 14 days after tumor transplantation. (A) Tumor sections were probed with anti-CD31 and anti–FSP-1 antibodies. Representative data are shown from 5 mice/group. Scale bar: 100 μm. Right, FSP-1 fluorescence intensity in CD31+ cells was quantified and expressed as percentage of the intensity in Metfl/fl mice treated with saline (n = 5, unpaired t test). (B) Tumor sections were fixed, probed with anti-CD31 and anti–NG-2 antibodies, and stained with Alexa Fluor 488– and 568–conjugated secondary IgGs (n = 3–5, representative data shown). Scale bar: 100 μm. (C) Mice were perfused i.v. with FITC-dextran. Tumors were excised, and the sections were imaged (n = 3–5, representative data shown). Scale bar: 50 μm. (D) Mice were injected with Hypoxyprobe-1 (pimonidazole HCl), and tumor sections were probed with antibody against pimonidazole adducts (n = 3–5, representative data shown). Scale bar: 100 μm.

Figure 9

Figure 9. A schematic model.

Cancer cell–derived HGF in the tumor microenvironment activates c-Met in EC, which in turn induces ETS-1–dependent MMP-14 expression, thereby inducing VE-cadherin cleavage and Endo-MT. Endo-MT generates abnormal vasculature and induces aberrant angiogenesis, leading to tumor progression and hypoxia-dependent tumor resistance.

References

    1. Carmeliet P, Jain RK. Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases. Nat Rev Drug Discov. 2011;10(6):417–427. doi: 10.1038/nrd3455. - DOI - PubMed
    1. Weis SM, Cheresh DA. Tumor angiogenesis: molecular pathways and therapeutic targets. Nat Med. 2011;17(11):1359–1370. doi: 10.1038/nm.2537. - DOI - PubMed
    1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–674. doi: 10.1016/j.cell.2011.02.013. - DOI - PubMed
    1. Stupp R, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352(10):987–996. doi: 10.1056/NEJMoa043330. - DOI - PubMed
    1. Huse JT, Holland EC. Targeting brain cancer: advances in the molecular pathology of malignant glioma and medulloblastoma. Nat Rev Cancer. 2010;10(5):319–331. doi: 10.1038/nrc2818. - DOI - PubMed

MeSH terms

Substances

LinkOut - more resources