PDGF-mediated mesenchymal transformation renders endothelial resistance to anti-VEGF treatment in glioblastoma - PubMed (original) (raw)

doi: 10.1038/s41467-018-05982-z.

Wenjuan Ma 1 3, Haineng Xu 1, Menggui Huang 1, Duo Zhang 1, Zhenqiang He 1 4, Lin Zhang 5, Steven Brem 6, Donald M O'Rourke 6, Yanqing Gong 7, Yonggao Mou 8, Zhenfeng Zhang 9, Yi Fan 10 11

Affiliations

PDGF-mediated mesenchymal transformation renders endothelial resistance to anti-VEGF treatment in glioblastoma

Tianrun Liu et al. Nat Commun. 2018.

Abstract

Angiogenesis is a hallmark of cancer. However, most malignant solid tumors exhibit robust resistance to current anti-angiogenic therapies that primarily target VEGF pathways. Here we report that endothelial-mesenchymal transformation induces glioblastoma (GBM) resistance to anti-angiogenic therapy by downregulating VEGFR-2 expression in tumor-associated endothelial cells (ECs). We show that VEGFR-2 expression is markedly reduced in human and mouse GBM ECs. Transcriptome analysis verifies reduced VEGFR-2 expression in ECs under GBM conditions and shows increased mesenchymal gene expression in these cells. Furthermore, we identify a PDGF/NF-κB/Snail axis that induces mesenchymal transformation and reduces VEGFR-2 expression in ECs. Finally, dual inhibition of VEGFR and PDGFR eliminates tumor-associated ECs and improves animal survival in GBM-bearing mice. Notably, EC-specific knockout of PDGFR-β sensitizes tumors to VEGF-neutralizing treatment. These findings reveal an endothelial plasticity-mediated mechanism that controls anti-angiogenic therapy resistance, and suggest that vascular de-transformation may offer promising opportunities for anti-vascular therapy in cancer.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Fig. 1

Tumor-associated ECs are resistant to anti-VEGF treatment and have diminished VEGFR-2 expression. a–c ECs were isolated from GBM tumors or peri-tumor tissues of human patients or normal brains. a, b Tumor ECs and normal brain microvascular ECs were treated with a 3 nM Ki8751 or b 10 μg/ml B20 antibody in VEGF-A-containing culture medium, and subjected to cell viability analysis (n = 3, mean ± SEM). c Cell lysates were immunoblotted. d, e The primary GBM in Ntv-a;_Ink4a-Arf_−/−;_Pten_−/−;LSL-Luc donor mice was induced by RCAS-mediated somatic gene transfer. Single-cell tumor suspension was injected into Rosa-LSL-tdTomato;Tie2-Cre mice. d Schematic approach. e Single-cell suspension isolated from normal brains or tumors were analyzed by flow cytometry. Left: representative sorting of CD11b− cells. Right: quantitative data (n = 4 mice, mean ± SEM). P value was determined by Student’s t test

Fig. 2

Fig. 2

PDGF induces downregulation of VEGFR-2 expression in ECs. a, b Normal human brain microvascular ECs (#1 and #2 from adult brain and #3 from fetal brain) were treated with glioma-conditioned medium (glioma-CM). RNA was isolated and subjected to transcriptome analysis by RNA deep sequencing (RNA-seq). Left, heat map for expression of VEGF receptors. Right, fold changes of VEGFR-1, VEGFR-2, and VEGFR-3 (n = 3, mean ± SEM). b Shown are FPKM values of FSP-1 (n = 3). c Normal brain ECs were treated with glioma-CM or control normal medium. Cell lysates were immunoblotted. d Gene set analysis of upregulated pathways/genes identified by RNA-seq in glioma-CM-treated ECs. e ECs were isolated from GBM tumors or peri-tumor tissues of human patients or normal brains. Cell lysates were immunoblotted. Note: the lyates were also immunoblotted in Fig. 1c, and the same blot for GAPDH was shown. f Normal brain ECs were treated with 100 ng/ml PDGF-AA, PDGF-AB, or PDGF-BB. Cell lysates were immunoblotted

Fig. 3

Fig. 3

PDGF is critical for glioma-CM-induced VEGFR-2 down-expression and EC resistance to anti-VEGF treatment. a, b Human brain microvascular ECs were treated with glioma-CM in the absence or presence of anti-PDGF-AA or anti-PDGF-BB neutralizing antibody or control IgG. a Cell lysates were immunoblotted. b RNA was isolated and analyzed by RT-PCR. Results were normalized with GAPDH levels (n = 3–5, mean ± SD). c Normal brain ECs were transfected with siRNAs targeting PDGFR-α, PDGFR-β, or control scrambled sequence, and treated with glioma-CM. Cell lysates were immunoblotted. d Human brain ECs were pretreated with 100 ng/ml PDGF-AB or PDGF-BB, followed by treatment with 3 nM Ki8751. Cell proliferation was determined (n = 3, mean ± SD). P values were determined by Student’s t test

Fig. 4

Fig. 4

PDGF-AB induces Endo-MT through NF-kB-mediated Snail expression in ECs. a Human brain microvacular ECs were treated with glioma-CM. RNA was isolated and subjected to RNA-seq analysis. Left, heat map for expression of EMT-related transcriptional factors. Right, fold change of these transcriptional factors (n = 3, mean ± SEM). b, c ECs were treated with 100 ng/ml PDGF-AA, PDGF-AB, and PDGF-BB. b Cell lysates were immunoblotted. c RNA was isolated and analyzed by RT-PCR. Results were normalized with GAPDH levels (n = 3, mean ± SEM). d ECs were transfected with siRNAs targeting Snail or control scrambled sequence, and treated with PDGF-AB. Cell lysates were immunoblotted. e ECs were transfected with siRNAs targeting Erg-1, NF-κB, or control scrambled sequence, and treated with PDGF-AB. Cell lysates were immunoblotted. f ECs were treated with PDGF-AB for 2 h. Cells were analyzed by immunofluorescence. g, h ECs were treated with PDGF-AB or control medium for 8 h. Nuclear extracts were immunoprecipitated with anti-NF-κB antibody or IgG, and subjected to ChIP analysis with primers #1 and #2. g DNA was resolved by agarose electrophoresis, and imaged. Shown are representative results with primer #2. The arrow indicates the amplified DNA in Snail promoter. h Quantitative PCR analysis (n = 3, mean ± SEM). i ECs were transfected with siRNAs targeting Snail or control scrambled sequence, 4 days after treatment with Ki8751, followed by cell viability analysis (n = 3, mean ± SD)

Fig. 5

Fig. 5

PDGF autocrine loop is critical for VEGFR-2 down-expression and anti-VEGF resistance in GBM-associated ECs. a GBM tumor-derived ECs were treated with control IgG or antibody against PDGF-AA or PDGF-BB. Cell lysates were immunoblotted. b, c GBM tumor-derived ECs were transfected with control scrambled siRNA or siRNA targeting PDGFR-α and PDGFR-β. b Cell lysates were immunoblotted. c Cell proliferation was determined (n = 3, mean ± SEM). d GBM tumor-derived ECs were treated with Ki8751 and crenolanib at different doses. Cell proliferation was determined 4 days after treatment. Inhibition rates were calculated and expressed as % of control cells

Fig. 6

Fig. 6

Dual inhibition of PDGFR and VEGFR-2 inhibits glioma growth and progression. The primary GBM was induced in Ntv-a;_Ink4a-Arf_−/−;_Pten_−/−;LSL-Luc donor mice by RCAS-mediated somatic gene transfer. Single-cell tumor suspension was implanted into recipient mice, followed by treatment. a Schematic approach. b Animal survival was monitored for 70 days after injection (n = 6–7 mice). MS median survival. P values were determined by log-rank tests. c Tumor growth was analyzed by whole-body bioluminescence imaging. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 32–38 (n = 3–6, mean ± SEM). P values were determined by Student’s t test. d Tumor sections were stained with H&E and imaged. Representative data are shown from 3–4 mice/group. Scale bar: 100 μm. e Tumor sections were immunostained with anti-CD31 antibody and imaged. Shown are representative images (n = 3–5 mice). Scale bar: 100 μm

Fig. 7

Fig. 7

Endothelial-specific deletion of PDGFR-b sensitizes glioma-associated ECs and tumors to anti-VEGF treatment. a–c _Tie2-Cre;Pdgfrb_fl/fl mice were generated by crossing Tie2-Cre mice with _Pdgfrb_fl/fl mice. a Schematic approach. b ECs were isolated from mouse aortas. Heart tissue and ECs were subjected to immunoblot analysis. c Mouse embryos were imaged (n = 5). d–h The primary GBM in Ntv-a;_Ink4a-Arf_−/−;_Pten_−/−;LSL-Luc donor mice was induced by RCAS-mediated somatic gene transfer. Single-cell tumor suspension was implanted into _Pdgfrb_fl/fl (WT) or Tie2-Cre;_Pdgfrb_fl/fl (PDGFR-β-ΔEC) recipient mice, followed by treatment with B20 antibody and IgG. d Schematic approach. e Animal survival was monitored for 60 days after injection (n = 7 mice). MS median survival. P values were determined by log-rank tests. f Tumor growth was analyzed by whole-body bioluminescence imaging. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 25–28 (n = 3–6 mice, mean ± SEM). g Tumor sections were stained with H&E dyes. Representative data are shown (n = 3–4 mice). Scale bar: 100 μm. h Tumor sections were immunostained with anti-CD31 antibody. Left, representative images. Right, quantitative analysis of CD31 fluorescence area (n = 4–5 mice, mean ± SEM). Scale bar: 100 μm. i Tumor sections were immunostained with anti-CD31 and anti-FSP-1 antibodies. Representative images are shown (n = 4–5 mice). Scale bar: 100 μm

Fig. 8

Fig. 8

A schematic model. PDGF in the tumor microenvironment activates PDGF receptor in ECs, which in turn induces NF-κB-dependent Snail expression, thereby inducing endothelial-mesenchymal transformation (Endo-MT). Snail binds to VEGFR-2 promoter and suppresses VEGFR-2 transcription, resulting in VEGFR-2 down-expression and Endo-MT, and eventually leading to anti-VEGF resistance in ECs. In addition, Endo-MT stimulates PDGF expression in ECs, potentially serving as an autocrine-mediated positive forward feedback loop that drives Endo-MT and EC resistance to anti-VEGF treatment

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