ARTICLE
Received 29 Jun 2015 | Accepted 9 Feb 2016 | Published 23 Mar 2016
Xiujuan Li1,*, Narendra Padhan1,*, Elisabet O. Sjstrm1, Francis P. Roche1, Chiara Testini1, Naoki Honkura1, Miguel Sinz-Jaspeado1, Emma Gordon1, Katie Bentley1,2, Andrew Philippides3, Vladimir Tolmachev1, Elisabetta Dejana1,4, Radu V. Stan5, Dietmar Vestweber6, Kurt Ballmer-Hofer7, Christer Betsholtz1,8, Kristian Pietras9, Leif Jansson10 & Lena Claesson-Welsh1
The specic role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2Y949F/Y949F leads to VEGFA-resistant endothelial adherens junctions and a block in
molecular extravasation. Vessels in Vegfr2Y949F/Y949F mice remain sensitive to inammatory
cytokines, and vascular morphology, blood pressure and ow parameters are normal. Tumour-bearing Vegfr2Y949F/Y949F mice display reduced vascular leakage and oedema,
improved response to chemotherapy and, importantly, reduced metastatic spread. The inammatory inltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms.
1 Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Science for Life Laboratory, Uppsala University, 751 85 Uppsala, Sweden.
2 Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215, USA. 3 Centre for Computational Neuroscience and Robotics, University of Sussex, Chichester 1 CI 104, Brighton BN1 9RH, UK. 4 c/o IFOM-IEO Campus, Via Adamello, 16, 20139 Milan, Italy.
5 Department of Pathology, Dartmouth College, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire 03756, USA. 6 Department of Vascular Cell Biology, Max Planck Institute for Molecular Biomedicine, Rntgenstra 20, 48149 Mnster, Germany. 7 Biomolecular Research, Molecular Cell Biology,
Paul-Scherrer Institute, 5232 Villigen-PSI, Switzerland. 8 Karolinska Institutet, Dept. Medical Biochemistry and Biophysics, Div. Vascular Biology, 17177 Stockholm, Sweden. 9 Translational Cancer Research, Medicon Village, Lund University, Building 404:A3, 22381 Lund, Sweden. 10 Department of Medical Cell Biology, Biomedical Center, Uppsala University, Box 571, 751 23 Uppsala, Sweden. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to L.C-W. (email: mailto:[email protected]
Web End [email protected] ).
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DOI: 10.1038/ncomms11017 OPEN
VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11017
was attenuated in the Vegfr2Y949F/Y949F mouse due to perturbed signalling through the TSAd/c-Src/VE-cadherin pathway resulting in VEGFA-resistant adherens junctions. The Y949F mutation however did not perturb other aspects of VEGFA-regulated vessel biology. The loss of VEGFA-regulated leakage was compatible with vascular development and a morphologically normal vasculature including the presence of fenestrated endothelium in adult organs. Blood ow and blood pressure changes in response to VEGFA were unaffected by the mutation. In cancer, the loss of VEGFA-regulated leakage was manifested as reduced tumour oedema, improved responsiveness to chemotherapy and suppressed metastatic spread due to an arrest in tumour cell intravasation.
ResultsVegfr2Y949F/Y949F vessels do not leak in response to VEGFA.Vegfr2Y949F/Y949F knock-in mice were created using Velocigene technology11 and used in this study after selection cassette removal, sequence verication and extensive backcrossing onto the C57Bl/6 background (Supplementary Fig. 1). The mutant mice were phenotypically normal and expressed similar levels of VEGFR2 protein in the vasculature as wild-type (WT) mice (see below). To determine the consequence of Vegfr2Y949F/Y949F mutation on VEGFA-induced vascular leakage in vivo, we employed two well-established assays, microsphere extra-vasation in the tracheal vasculature and Miles extravasation assay in the dermis. To induce microsphere extravasation, VEGFA or vehicle was administered by tail vein injection together with uorescent 30-nm microspheres. VEGFA-induced extravasation of microspheres, lodged beneath the basement membrane in postcapillary venules, was signicantly reduced at 60 min of circulation in the Vegfr2Y949F/Y949F trachea compared with WT (Fig. 1a,b). In contrast, tail vein-administered histamine induced similar levels of microsphere extravasation in WT and Vegfr2Y949F/Y949F trachea venules (Fig. 1c,d). Moreover, VEGFA induced a signicant increase of Evans blue leakage in the skin (Miles assay) in the WT but not in the Vegfr2Y949F/Y949F mouse (Fig. 1e).
We have previously shown that the phosphorylated Y949 site in VEGFR2 serves as a binding site for Src homology 2 (SH2) domain of TSAd8, which in turn binds the SH3 domain of c-Src. c-Src is known to regulate VE-cadherin and adherens junctions integrity12. We veried VEGFA-induced VEGFR2/TSAd complex formation in the WT and lack of TSAd binding to mutant
Figure 1 | Arrest in VEGFA-induced vascular leakage in Vegfr2Y949F/Y949F mice. (a) Tracheal vessel leakage in WT (left) and Vegfr2Y949F/Y949F (Y949F;
right) mice after tail vein injection of VEGFA or PBS combined with 30 nm uorescent microspheres (green). Arrows: microsphere leakage from CD31 vessels (red). Scale bar, 50 mm. (b) Microsphere area per CD31-positive area fraction in VEGFA/PBS samples from a. n 3 mice per genotype. Two-way
ANOVA: P(treatment)o0.0001; P(genotype) 0.001; P(interaction) 0.0008. (c) Tracheal vessel leakage in response to tail vein-injected histamine
(as in a), in WTand Vegfr2Y949F/Y949F (Y949F) mice. (d) Microsphere area per CD31-positive area fraction in histamine/PBS samples from c. n 36 mice
per genotype. Two-way ANOVA: P(treatment)o0.0001; P(genotype) 0.84; P(interaction) 0.80. (e) Evans blue extravasation induced by VEGFA/PBS
in the dermis of WT and Vegfr2Y949F/Y949F (Y949F) mice. Values were normalized to tissue weight. n 3 mice per genotype. Two-way ANOVA:
P(treatment) 0.0053; P(genotype) 0.0206; P(interaction) 0.0482. (f) VEGFR2/TSAd complex formation in isolated mouse lung endothelial cells
from WT and Vegfr2Y949F/Y949F (Y949F) mice, treated or not with VEGFA. Immunoprecipitation (IP) for VEGFR2 and immunoblotting for TSAd and
VEGFR2. Total lysates were immunoblotted for TSAd and tubulin. Positive control, total lysate. Molecular weight markers to the right. (g) VE-cadherin immunostaining (yellowblack heatmap) on trachea from WT and Vegfr2Y949F/Y949F (Y949F) mice, tail vein-injected with VEGFA/PBS and microspheres
(pinkred heatmap). Images were rendered using structured illumination microscopy. Note VE-cadherin rearrangement in VEGFA-injected WT trachea. Scale bar, 10 mm. (h) VE-cadherin fragments per mm vessel length from g. n 36 individual tissues per genotype. Total vessel length assessed: WT/PBS,
236 mm; WT/VEGFA, 308 mm; Y949F/PBS, 150 mm; and Y949F/VEGFA, 145 mm. Two-way ANOVA: P(treatment) 0.0022; P(genotype) 0.0065;
P(interaction) 0.0164. (i) VE-cadherin immunostaining (red) on endothelial cells isolated from WTand Vegfr2Y949F/Y949F (Y949F) mouse lungs and then
treated or not with VEGFA (100 ng ml 1). Hoechst 33342 (blue) staining shows nuclei. Scale bar, 20 mm. (j) VE-cadherin-positive staining per cell; samples generated as in i. n 30 cells per condition. Two-way ANOVA: P(treatment) 0.0189; P(genotype) 0.099; P(interaction) 0.0741. Data presented as
means.e.m. Two-way ANOVA with Tukeys post hoc test, *Po0.05, **Po0.01, ***Po0.001, ****Po0.0001. Experiments were performed at least three independent times.
Cancer is characterized by a dysfunctional and morphologically abnormal vasculature with elevated permeability and sparse pericyte and basement membrane coating,
resulting in unstable vessels and poor blood ow1. The dismal condition of the tumour vasculature is attributed to the hypoxic tumour micro-environment and the accompanying inammation resulting in the production of a broad range of growth modulatory factors including vascular endothelial growth factors (VEGFs), in particular VEGFA (ref. 2). VEGFA causes increased vascular leakage by disruption of adherens junctions created through homophilic interactions between vascular endothelial (VE)-cadherin molecules expressed on adjacent endothelial cells3. Vessel normalization by titrating an appropriate level of anti-VEGFA therapy improves the efcacy of irradiation and chemotherapy4. However, sustained anti-VEGFA therapy deteriorates vessel function and may cause increased hypoxia, increased local tumour cell invasion and increased metastatic propensity (for a review, see ref. 5). The role of VEGFA-induced permeability compared with VEGFA-regulated migration, survival and proliferation of endothelial cells is unknown. Moreover, the signal transduction pathways induced by VEGFA to promote increased vascular permeability have remained unidentied.
VEGFA exerts its effects via two endothelial cell receptor tyrosine kinases denoted VEGFR1 and VEGFR2 (ref. 6). VEGFR1 binds and neutralizes VEGFA, thereby exerting a negative regulatory effect on endothelial cells, while VEGFR2 is essential in all known VEGFA biology6. VEGFR2 becomes activated and phosphorylated on tyrosine residues in response to VEGFA: Y949, Y1052, Y1057, Y1173 and Y1212 (mouse sequence numbers)7. The Y949 phosphosite in VEGFR2 (Y951 in human VEGFR2) presents a specic binding site for the T cell-specic adaptor (TSAd). TSAd is implicated in VEGFA-induced permeability, by regulating VEGFR2-dependent c-Src signalling at endothelial cell junctions8. The Y1052/1057 residues, located on the tyrosine kinase activation loop, are required for full kinase activity (see ref. 6 and references therein). The phosphorylated Y1173 binds phospholipase Cg, of importance for endothelial
ERK1/2 pathway activation9. A phenylalanine knock-in mouse Vegfr2Y1173F/Y1173F is embryonically lethal due to arrested endothelial cell development10. The Vegfr2Y1212F/Y1212F mouse lacks a developmental phenotype on a mixed background10.
The aim of the current study was to examine the consequence of specic suppression of VEGFA-induced permeability on cancer progression. VEGFA-induced molecular extravasation
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VEGFR2-Y949F, by immunoblotting on isolated endothelial cells from WT and Vegfr2Y949F/Y949F mouse lungs (Fig. 1f).
We next analysed the consequence of the VEGFR2-Y949F mutation of VE-cadherin stability. Immunostaining for VE-cadherin visualised continuous cell borders in WT and Vegfr2Y949F/Y949F tracheal vessels, analysed using structured illumination microscopy imaging (Fig. 1g). In response to
VEGFA, the WT tracheal vessels, but not Vegfr2Y949F/Y949F vessels, showed a signicant increase in fragmented VE-cadherin staining (Fig. 1g, quantied in h) indicative of VE-cadherin internalization and breakdown of the adherens junctions causing transient vascular leakage13. The difference in VE-cadherin dynamics between the genotypes was validated using endothelial cells isolated from mouse lungs. VEGFA promoted
a
WT Y949F
b
VEGF
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Microsphere per CD31+ area
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IP:VEGFR2 from isolated endothelial cells from mouse lungs
80 ** *
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VEGF
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Positive control
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TSAd / VEGFR2 ratio
kDa
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225 52
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IB
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VEGFR2
Input TSAd
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WT PBS WT VEGF Y949F PBS Y949F VEGF
VE-cadherin Microsphere
VE-cadherin fragments
per m
Quantification g
*** ***
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WT PBS WT VEGF Y949F PBS Y949F VEGF VE-cadherin DAPI
Quantification i
* NS
VE-cadherin area
per cell (%)
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WT
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VE-cadherin junction disintegration in the WT cells but not in the Vegfr2Y949F/Y949F cells, resulting in a signicantly increased VE-cadherin-positive staining area in the VEGFA-treated WT cells (Fig. 1i,j).
Vegfr2Y949F/Y949F vessel characteristics. The Vegfr2Y949F/Y949F mice showed no developmental vascular abnormality, for
example, in the embryonic day 11.5 (E11.5) hindbrain (Supplementary Fig. 2ad) and were born at the expected Mendelian ratios. There was also no apparent effect of the mutation on the development and organization of lymphatics (see Supplementary Fig. 2e for tracheal lymphatics). The postnatal retinal vasculature develops in a strictly regulated, VEGFA-dependent manner to establish the junctional barrier14.
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IB4 ERG
Cells with filopodia
No. of sprouts per field
Vascular area
Outgrowth
Radial expansion per field
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Distribution of patches
VE-cadherin classification key
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Expression of endothelial genes
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Desmin
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Cldn5
Pecam1
Cdh5
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Podxl
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Nos3
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There was no difference in retinal vascular development between WT and Vegfr2Y949F/Y949F mice as judged by the overall morphology of the vascular network, the abundance of endothelial tip cells and lopodia, and the total vascular area (Fig. 2ad). Thus, development of the retinal vasculature does not depend on VEGFR2-Y949 signalling. However, the morphology of the VE-cadherin immunostaining differed between the two genotypes. The VE-cadherin pattern at individual junctions was evaluated using established image analysis software15, classifying junctions in a graded scale from activethat is, being irregular/ serrated with vesicular/diffuse appearanceto inactive ones, presenting a straighter and less vesicular morphology (Fig. 2e, see Fig. 2f,g for quantication and schematic outline). By this criterion, the WT retinal vasculature displayed a signicantly higher prevalence of active adherens junctions compared with the Vegfr2Y949F/Y949F retinal vasculature (Fig. 2f; active junction patches was 58.9% (WT) versus 48.1% (Vegfr2Y949F/Y949F);
P 0.0367, calculated using median% in one-tailed Wilcoxon
rank sum test.
Still, the enhanced barrier morphology in the VEGFR2-Y949F mutant did not interfere with overall vascular morphology or physiology. The adult vascular ultramorphology of the kidney glomeruli and peritubular capillaries (Fig. 2h) and the pancreas (Supplementary Fig. 2f) was normal in the Vegfr2Y949F/Y949F mouse. Moreover, expression of plasmalemma vesicle-associated protein-1 (PV1), which is required for the formation of diaphragms covering endothelial fenestrae in vessels of endocrine organs16 was not affected by the VEGFR2-Y949F mutation (Fig. 2i).
To exclude the possibility that vascular leakage arrest in the Vegfr2Y949F/Y949F mouse was inuenced by blood pressure/ow parameters, a series of in vivo tests were performed. The resting blood pressure in the descending aorta was monitored continuously while infusing VEGFA or phosphate-buffered saline (PBS) by tail vein injection. VEGFA, but not PBS, caused a signicant drop in blood pressure as an indication of vessel dilation, in both WT and Vegfr2Y949F/Y949F mice (Fig. 2j). Moreover, blood parameters including arterial gases, oxygen saturation, serum Na /K levels, as well as the haematocrit/
haemoglobin levels were similar in the WT and Y949F conditions (Table 1). Tail vein injection of lectin showed that the WT and Vegfr2Y949F/Y949F tracheal vessels were perfused to the same extent (Supplementary Fig. 2g), supporting the notion that the leakage arrest in the mutant mouse vasculature was not due to altered circulation. Pericyte morphology (Fig. 2k,l) and basement membrane coating (Fig. 2m,n) were similar between the genotypes. Moreover, RNAseq analyses did not reveal major differences in expression levels of key endothelial transcripts between the WT and the Vegfr2Y949F/Y949F condition (Fig. 2o).
Reduced Vegfr2Y949F/Y949F glioblastoma vessel leakage. The integrity of the central nervous system vasculature is tightly controlled by the bloodbrain barrier, in which endothelial junctions are particularly tight17. We asked whether VEGFA-regulated vascular leakage has an impact on the growth and vascular permeability of brain tumours. The GL261 glioma model has many features reminiscent of human glioma, including abnormal vasculature, high VEGFA production and oedema18. GL261 glioma cells were injected in the right medial striatum of WT and Vegfr2Y949F/Y949F mice. There was no effect on tumour growth at any examined time point after cell inoculation (day 7 (D7), D14, D18 and D21; Fig. 3a shows tumour volume at D18). There was also no difference in the extent of perfused vessel
Table 1 | Haemodynamics in WT and Vegfr2Y949F/Y949F mice.
Strain WT (n 9)
Y949F (n 8)
P value
pH 7.290.01 7.320.02 0.288 Arterial pCO2 (kPa) 5.80.3 5.50.3 0.596
Arterial pO2 (kPa) 13.10.9 14.70.6 0.167
Base excess (mmol l 1) 5.80.6 4.61.0 0.341
Arterial HCO3 (mmol l 1) 20.70.7 21.31.1 0.661 Arterial oxygen saturation (%) 95.61.0 97.50.4 0.185
S-Na (mmol l 1) 144.80.6 143.20.5 0.09 S-K (mmol l 1) 5.10.2 5.20.2 0.757
Haematocrit (%) 32.31.1 33.01.6 0.73 Haemoglobin (g l 1) 1104 1125 0.732
Blood ow values*Pancreatic
Whole gland 1.760.36 1.420.22 0.458 Caput 2.100.48 1.470.20 0.532 Cauda 1.520.32 1.380.24 0.735 Duodenal 4.571.21 3.600.70 0.514 Colonic 1.510.24 1.480.47 0.361 Lung 0.150.07 0.210.09 0.665 Kidney 7.151.65 5.691.28 0.503 CNS 1.070.18 1.640.48 0.47 Fat 0.0130.004 0.0080.003 0.768
CNS, central nervous system. Values are meanss.e.m. *ml min 1 g organ weight.
Figure 2 | Vegfr2Y949F/Y949F vessel parameters. (a) Isolectin-B4 (IB4, red) and ERG (green) immunostaining on WT and Vegfr2Y949F/Y949F (Y949F) P6
retinal vasculature. Scale bar, 100 mm. (b) Tip cells numbers. n 34 retinas per genotype. (c) IB4-positive vessel area normalized to WT. n 34 retinas per
genotype. (d) Radial vessel outgrowth normalized to WT. n 45 retinas per genotype. (e) Heatmap of VE-cadherin morphology in WTand Vegfr2Y949F/Y949F
(Y949F) P6 retinas. Scale bar, 50 mm. (f) Retinal images in e processed for VE-cadherin morphology, lying within the vascular mask, into patches of 100 100 pixels ( 63, n 6 per group). The VE-cadherin morphology in each patch was hand-classied according to the classication key in g. (g)
Adherens junctions classication from active (red; irregular/serrated morphology with diffuse/vesicular regions) to inactive (blue; straighter morphology with less vesicular staining). (h) Transmission electron microscopy analysis of kidney glomerulus and peritubular capillary (cap) show normal appearance of junctions (red arrows) and fenestrae (insets) in the two genotypes. (i) PV1 protein immunoblot in lung lysates after injection of PBS (P) or VEGFA (V) and circulation for 110 min (P1; injection of PBS and 1 min circulation and so on). GAPDH; equal loading. Molecular weight markers to the right. (j). Blood pressure 10 min after saline or VEGFA infusion, monitored in the ascending aorta. Values (positive or negative) show diastolic blood pressure (BP) normalizedto BP before VEGFA/PBS injection (70 mm Hg; NS between genotypes). n 68 mice per genotype. Students t-test, *Po0.05. Two-way ANOVA:
P(treatment) 0.0001; P(genotype) 0.738; P(interaction) 0.5321. (k) CD31-positive (red) tracheal endothelium and desmin-positive (green) pericytes.
Scale bar; 50 mm. (l) Pericyte area per total area from k. n 4 mice per genotype. (m) CD31-positive (red) tracheal endothelium and collagen IV-positive
(green) basement membrane. Scale bar; 50 mm. (n) Collagen IV area per total area in m. n 3 mice per genotype. (o) RNAseq analysis of total lung polyA
RNA. Data for selected endothelial genes as log10-scaled fold-change based on the frequency of normalized expression. For Vegfr2 (KDR) the Y949F mutant showed a 0.24 (DESeq2log2) fold decrease compared with WT. Data presented as means.e.m. Students t-test (bd) and Wilcoxon rank test (f); NS, not
signicant. Experiments were performed three independent times or performed once with at least three independent data sets (j,o).
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density (Fig. 3b). However, leakage of tail vein-injected cadaverine-Alexa-555, a small tracer that normally does not cross the bloodbrain barrier19, was reduced in the Vegfr2Y949F/Y949F gliomas compared with WT at D18, both intratumourally and in the tumour periphery (0500 mm peritumourally; Fig. 3c). The glioma vasculature in the WT was more disorganized than in the Vegfr2Y949F/Y949F (Fig. 3d). Hotspots of uorescent lectin leaking from perfused vessels (Fig. 3d, boxed area) correlated with high tumour uptake of Cadaverine-555 (Fig. 3e) in the WT compared with the mutant.
RIP1-TAg2:Vegfr2Y949F/Y949F leakage and metastatic spread. The consequence of the stabilized endothelial junctions in
Vegfr2Y949F/Y949F mice during tumour progression was further explored in the RIP1-TAg2 model. b-cells in the islets of Langerhans in RIP1-TAg2 mice express SV40 T antigen controlled by the rat insulin promoter, giving rise to multiple spontaneous pancreatic neuroendocrine tumours20 that seed metastases in the liver21. While there was no difference in the total tumour volume between the RIP1-TAg2:WT and RIP1-TAg2:Vegfr2Y949F/Y949F genotypes (Fig. 4a), the number of angiogenic islets, which are small lesions representing an early stage in tumour development, was signicantly increased in RIP1-TAg2:Vegfr2Y949F/Y949F mice (Fig. 4b). Conversely, the RIP1-TAg2:Vegfr2Y949F/Y949F presented with fewer large (45 mm) neoplastic islet tumours (Fig. 4c).
Tumour cells were found in close proximity to the vasculature in the tumour periphery suggesting metastatic spread of the
a
b
c
Glioma tumour volume
Cadaverine accumulation
Vessel fraction per tumour area
0.08
Perfused vessel density
0.06
0.04
NS
NS
Fluorescence intensity (% of Ctrl)
50
Tumour volume (mm3 )
120 Intratumoural Peritumoural
* **
40
80
30
20
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Lectin Cadaverine
e
Figure 3 | Reduced glioblastoma permeability in Vegfr2Y949F/Y949F mice. (a) GL261 glioma volumes at D18, estimated by measuring all tumour-
containing sections in series throughout the brain of WT and Vegfr2Y949F/Y949F (Y949F) mice. n 1314 mice per genotype. (b) Lectin-positive, perfused
vessel density (area fraction per tumour area) at D18. n 1214 mice per genotype. (c) Cadaverine-555 uorescence intensity units estimated within the
tumour (intratumoural) and at a 0500-mm rim around the tumour border (peritumoural), Y949F normalized to WT. n 811 mice per genotype.
(d) GL261 lectin-perfused vessels (lectin in green). Boxed area, typical point of leakage/disrupted barrier in the WT. (e) Cadaverine-555 (red) extravasation in WT and Y949F GL261 gliomas. Scale bar, 100 mm (d) and 50 mm (e). Data are presented as means.e.m. Students t-test, *Po0.05, **Po0.01. Experiments were performed three independent times. NS, not signicant.
Figure 4 | Reduced RIP1-TAg2 vascular leakage and metastatic spread in Vegfr2Y949F/Y949F. (a) RIP1-TAg2 WTand Vegfr2Y949F/Y949F insulinoma tumour
volumes at week 14. n 1921 mice per genotype. (b) Angiogenic islet numbers at week 14. n 1416 mice per genotype. (c) Tumour size distribution (15
and 45 mm diameter), week 14. n 1719 mice per genotype. (d) Tumour cells (SV40 T antigen; green) and vessels (CD31; red) in RIP1-TAg2
insulinomas (left), Arrows; individual tumour cells associated with vessels. Scale bar, 100 mm. SV40 Tantigen (green; upper) and haematoxylin-eosin (HE; lower) metastatic liver lesion (right). Scale bars, 50 mm. Number of liver metastases; mean per section, 30 sections per liver. n 1117 mice per genotype.
(e) Leakage of 30-nm microspheres (green; arrows) from VE-cadherin tumour vessels (red). Scale bar, 50 mm. (f) Microspheres area per VE-cadherin area from e. n 1011 tumours per genotype. (g,h) VE-cadherin (red) and podocalyxin (green) RIP1-TAg2 tumour vasculature. Lower panels, magnication of
boxed areas in upper panels. Arrows indicate typical disorganized podocalyxin in WT tumour vasculature. Scale bars, 50 mm (g), 5 mm (h). (i) VE-cadherin area, % of total area from g. n 1517 tumours per genotype. (j) Podocalyxin area, % of total area from g. n 1517 tumours per genotype. Data shown as
means.e.m. Students t-test, *Po0.05, **Po0.01, NS, not signicant. Data are pooled from three independent studies.
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11017 ARTICLE
insulinoma (Fig. 4d). Examination of livers showed that the number of SV40 T-positive liver metastases in the RIP1-TAg2:WT was signicantly higher than in RIP1-TAg2:Vegfr2Y949F/Y949F
mice at week 14 (Fig. 4d). We hypothesized that reduced metastatic spread in RIP1-TAg2:Vegfr2Y949F/Y949F mice could correlate with reduced vascular leakage. Tail vein-injected
a
b
c
Pancreatic tumour volume
Angiogenic islets
Tumour size distribution
15 mm
NS
NS
200
25 *
No. of angiogenic
islets per pancreas
8
*
Tumour volume (mm)
20
6
100
Tumour no. per pancreas
0
150
15
4
10
50
2
5
0
0
WT Y949F
WT Y949F
WT WT Y949F
Y949F
> 5 mm
d
Liver metastases
SV40 CD31 SV40 DAPI
HE
10 **
Liver metastasis (mean)
8
6
4
2
0
WT Y949F
e
WT Y949F f
Microsphere area
Microsphere per VE-cadherinarea
VE-cadherin Microsphere
0.20
**
0.15
0.10
0.05
0.00
WT Y949F
g
WT Y949F
i
VE-cadherin Podocalyxin
VE-cadherin area
15 *
WT Y949F
VE-cadherin area (%)
10
5
0
h
WT Y949F
j
Podocalyxin area
30
20
10
0
*
Podocalayxin area (%)
WT Y949F
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11017
a Perfusion
b
B16F10 tumour volume
CD31+ area
c
D12
D18
D12
D18
NS NS
1.5
*
Tumour volume (mm3 )
2,500
2,000
1,500
WT Y949F
12 10
Lectin+ per CD31+ area
0.0
5
CD31+ area
NS
4
1.0
1,000
2
500
0 6 8 10 12 14 16 18
0.5
0
Days after implantation
WT Y949F
WT Y949F
WT Y949F WT
NS
Y949F
d
e
WT Y949F
Fibrinogen leakage
WT Y949F WT Y949F
D12
D18
Fibrinogen+ per CD31+ area
2
CD31
Fibrinogen
8
0
* NS
6
4
f
Tumour water content
Y949F
g
D12
600 *
Tumour volume (mm3 )
D18
NS
Chemotherapy
NS
100
% Water content
**
300
280
90
200
80
100
70
60 0 WT Y949F
0
Control TMZ
Y949F D12
Control TMZ
WT
WT D12
h
i
Lung metastases
800
B16F10 ear tumour volume
**
WT Y949F
Tumour volume (mm3 )
600
400
Lung metastases per lobe
15
10
5
0
NS
200
0 6 8 10 12 14
16 Days after implantation
WT Y949F
Figure 5 | Reduced B16F10 vascular leakage and metastatic spread in Vegfr2Y949F/Y949F. (a) Subcutaneous B16F10 tumour volumes for WT (blue) and
Vegfr2Y949F/Y949F (Y949F; magenta) mice at different days after inoculation. n 1415 mice per genotype. Repeated measures ANOVA: P(time)o0.0001;
P(genotype) NS. (b) CD31 area per eld in B16F10 tumours from WTand Y949F mice. n 21 mice per genotype at D12 and 1116 mice per genotype at
D18. (c) Area of tomato lectin-perfused vessels normalized to CD31 area. n 21 mice per genotype, D12; and 1116 mice per genotype at D18. *Po0.05.
(d) Tumour vasculature (upper panels; CD31 vessels; white) and perivascular brinogen deposition (lower panels, heatmap from red, high, to blue, low) in B16F10 tumours from WT and Vegfr2Y949F/Y949F mice at D12. Scale bars, 50 mm. (e) Fibrinogen area normalized to CD31 area. n 2227 mice per
genotype at D12 and 1314 mice per genotype at D18. Students t-test, P 0.0310. (f) Oedema in B16F10 tumours estimated by weighing tumours before
and after drying. Data show (wet weight dry weight)/wet weight in % at D12 and D18. n 16-18 mice per genotype at D12 and 1924 mice per genotype
at D18. Students t-test, P 0.0385. (g) Tumour volumes at D12 of TMZ or vehicle (dimethylsulfoxide)-treated mice with B16F10 tumours, receiving
treatment between D4 and D8 after inoculation. n 1215 mice per group. KruskalWallis test, P 0.0289. (h) B16F10 tumour volumes in mouse ear.
n 1213 mice per genotype. Repeated measures ANOVA: P(time)o0.0001; P(genotype) NS. (i) Spontaneous B16F10 lung metastasis spreading to the
lungs from primary B16F10 tumours in the ear. dsred-B16F10 metastases in four longitudinal sections of each left main lung lobe were counted. n 12 mice
per genotype. MannWhitney U, P 0.0028. Data shown as means.e.m. Dashed lines in b,c,e and f indicate that data sets from D12 and D18 were not
compared statistically. *Po0.05, **Po0.01, NS, not signicant. Experiments were performed three independent times (ae,hj) or two times (f,g), and data were pooled.
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11017 ARTICLE
microspheres leaked from tumour vessels to a higher extent in RIP1-TAg2:WT than in RIP1-TAg2:Vegfr2Y949F/Y949F tumours (Fig. 4e,f). In the RIP1-TAg2:Vegfr2Y949F/Y949F tumour vasculature, VE-cadherin was preferentially localized at endothelial junctions and the VE-cadherin area was lower than in the WT (Fig. 4gi). Moreover, staining for the sialomucin podocalyxin decorated the luminal endothelial surface in the RIP1-TAg2:Vegfr2Y949F/Y949F tumour vasculature while RIP1-TAg2:WT tumours showed a chaotic podocalyxin staining pattern suggestive of altered apicalbasal (luminalabluminal) endothelial polarity (Fig. 4g,h,j) and therefore, disturbed vessel organization. The extent of vessel perfusion, pericyte coating and inammatory cell inltration (that is, CD45, F4/80 and macrophage mannose receptor (MMR)-positive cells did, however, not differ between RIP1-TAg2:WT and RIP1-TAg2:Vegfr2Y949F/Y949F tumours at week 14 (Supplementary Fig. 3ae).
B16F10 vessel leakage and metastatic spread. To further test the effect of the VEGFR2-Y949F mutation on vascular integrity in cancer, we chose to study subcutaneous B16F10 melanoma22. B16F10 tumour growth in the back skin was monitored continuously showing similar growth curves for the two genotypes (Fig. 5a). To monitor dynamic changes in tumour and vascular parameters, tumours were collected at two different time points after inoculation, at D12 (average tumour volume 200 mm3) and at D18 (average tumour volume 1.5 cm3) from WT and Vegfr2Y949F/Y949F mice. Vascular area (Fig. 5b) and pericyte density (Supplementary Fig. 3f) were similar in WT and Vegfr2Y949F/Y949F B16F10 tumours at D12 and D18. Vessel functionality as assessed by lectin perfusion was more efcient in the Vegfr2Y949F/Y949F tumour vasculature at D12 (Fig. 5c). At D18, vessel perfusion had improved in the WT and was similar to that of Vegfr2Y949F/Y949F tumours. In spite of the improved perfusion, necrosis in the tumour was similar in the two genotypes (Supplementary Fig. 3g). Importantly, there were no differences in inltration of F4/80,
CD45 and MMR-positive inammatory leukocytes in the tumours at D12 (Supplementary Fig. 3hj).
Vessel leakage in B16F10 tumours, determined by estimating the deposition of perivascular brinogen (Fig. 5d; quantied in e), was signicantly lower in Vegfr2Y949F/Y949F tumours compared with WT tumours at D12. At D18, brinogen deposition was reduced in the WT tumours and similar between the two genotypes. Vascular leakage in tumours leads to oedema, which was estimated by weighing tumours before and after drying. The water content of Vegfr2Y949F/Y949F tumours was signicantly lower than in WT tumours at D12 (Fig. 5f) but not at D18. Altogether, these data indicate that the block in VEGFR2 pY949 signalling constrained vascular leakage and oedema in the tumour up to a critical point in tumour growth, occurring around D12. Thereafter, leakage and oedema were reduced irrespective of genotype, possibly due to an overall dysfunctional tumour circulation.
To test the effect of chemotherapy on tumours exhibiting different extents of vessel leakage and oedema, B16F10 tumour-bearing mice were treated with a subtherapeutic dose of temozolomide (TMZ) or vehicle in one cycle for 5 consecutive days. Tumour growth in WT mice was resistant to the TMZ treatment at the given dose. In contrast, TMZ-treated Vegfr2Y949F/Y949F mice had smaller tumours than WT at D12 (Fig. 5g) but not at D18 (Supplementary Fig. 4a). Thus, the VEGFA-resistant vessel barrier phenotype in the Vegfr2Y949F/Y949F tumours correlated with an enhanced drug effect at D12, probably due to the lower oedema, and hence a lower interstitial tumour pressure, at this stage.
To study B16F10 metastasis, dsRed-B16F10 cells were inoculated intradermally in the ear. Tumour growth rate is constrained in the ear dermis, which we found correlated with an increased propensity to metastasize compared with when tumours grow unconstrained in the back skin. The primary tumour growth rate was similar between Vegfr2Y949F/Y949F and WT mice (Fig. 5h). Metastatic spread from the primary dsRed-B16F10 tumour to the lungs was however signicantly reduced in the Vegfr2Y949F/Y949F mice (Fig. 5i). There was no correlation between the end point weight of the primary tumour and the number of metastatic foci (Supplementary Fig. 4b). We further tested the capacity of tail vein-injected dsRed-B16F10 tumour cells to colonize lungs and found no difference in lodging frequency between the genotypes (Supplementary Fig. 4c).
In vivo signalling in the Vegfr2Y949F/Y949F endothelium. To decipher signalling regulating VEGFA-induced vascular leakage in vivo, mice were subjected to tail vein injection of VEGFA or vehicle, and lungs were collected after different time periods of circulation (160 min). Lungs were chosen since they consist at least 50% of endothelial cells. Moreover, we used the two-tracer technique23 to show that WT lung endothelial cells respond to VEGFA with increased transvascular leakage of iodinated albumin in vivo. The leakage rate in WT mice increased2.5-fold after VEGFA injection in the tail vein, compared with vehicle (ow rate in response to VEGFA, 0.8590.114 versus vehicle, 0.3340.126 ml min 1 g 1; s.e.m., P 0.0278, n 34
mice per condition).
VEGFA induced a very rapid and strong phosphorylation of VEGFR2 at Y1173 in WT and to a lower relative level in the Vegfr2Y949F/Y949F lung endothelium (Fig. 6a). Phosphorylation at Y949 was also high at 1 min after VEGFA injection in the WT, whereas it was absent in the Vegfr2Y949F/Y949F vasculature (Supplementary Fig. 5a). The lower pY1173 accumulation in the Vegfr2Y949F/Y949F could be due to the more extensive complex formation with the vascular endothelial protein tyrosine phosphatase (VEPTP). As shown in Supplementary Fig. 5b, there was a signicantly higher extent of VEPTP/VEGFR2 complex formation in the Vegfr2Y949F/Y949F immunoprecipitates compared with WT VEGFR2 (two-way analysis of variance (ANOVA), genotype effect: P 0.0037). VEPTP has been shown to restrict
VEGFR2 tyrosine phosphorylation24.
Several signalling pathways regulate vascular permeability and leakage, including VEGFA-induced c-Src signalling8,25 and histamine-induced phosphoinositide 3 kinase/Akt signalling26, which both induce phosphorylation of VE-cadherin and VE-cadherin internalization. We examined induction of pY658 VE-cadherin by immunoblotting of VEGFR2 immunoprecipitates from mouse lungs. We choose to study VE-cadherin in complex with VEGFR2 to monitor direct communication between the two. In the WT, the levels of pY658 VE-cadherin were already high in the PBS-injected mice in agreement with previous reports27, and remained high initially after injection of VEGFA, followed by a gradual decrease possibly due to the action of phosphatases, or to internalization and degradation of VE-cadherin (Fig. 6a). In the Vegfr2Y949F/Y949F-derived samples, the pVE-cadherin level was low in the PBS condition and there was no induction with VEGFA injection (Fig. 6a).
Tyrosine phosphorylation of c-Src was difcult to detect in the VEGFR2 immunoprecipitates from lung lysate and was therefore examined in total lung lysates (Fig. 6b). In the WT samples, we detected an increased accumulation of pY418 c-Src only at the late time point, in agreement with previous data where c-Src activation was detected in lung lysates 20 min after tail vein injection of VEGFA8. In the Vegfr2Y949F/Y949F mouse lungs,
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a
IP:VEGFR2
15
VEGFR2 pY1173 / VEGFR2
WT Y949F
***
****
**
VEGFR2 pY1173
VEGFR2
WT Y949FP1 P2 P5 V1 V2 V5 V10 V15 V60 P1 P2 P5 V1 V2 V5 V10 V15 V60 kDa
225
225
Ratio VEGFR2 pY1173 /
VEGFR2
IB
10
5
0 P15 V12 V515 V60
VE-cad pY658 / VE-cad
1.5
0.0
WT Y949FP1 P2 P5 V1 V2 V5 V10 V15 V60 P1 P2 P5 V1 V2 V5 V10 V15 V60 kDa
150
150
Ratio VE-cad pY658 /
VE-cad
IB
VE-cad pY658
VE-cad
1.0
*
0.5
P15 V12 V515 V60
b
TLL
2.0 Src pY418 / Src
P15 V12 V515 V60
Ratio Src pY418 / Src
0.5
WT Y949F
*
WT Y949FP1 P2 P5 V1 V2 V5 V10 V15 V60 P1 P2 P5 V1 V2 V5 V10 V15 V60 kDa
52
52
kDa
55
55
1.5
0.0
IB
Src pY418
1.0
Src
P15 V12 V515 V60
Akt pT308 / Akt
1.5
0.0
Ratio Akt pT308 / Akt
WT Y949FP1 P2 P5 V1 V2 V5 V10 V15 V60 P1 P2 P5 V1 V2 V5 V10 V15 V60
IB
1.0
Akt pT308
Akt
0.5
c d e
IP: VEGFR2, VEGFR2 activity
*
IP: VEGFR2, Src activity
*
IP: VEGFR2, VEPTP complex
**
D12
D18
D12
WT Y949F
D18
D12
WT Y949F
D18
Ratio pY 1173 VEGFR2 / VEGFR2
2.0
4.0
Ratio pY418 Src / Src
0.8
Ratio VEPTP / VEGFR2
1.4
0.8
3.0
0.6
0.6
2.0
0.4
0.4
1.0
0.2
0.2
0.0
0.0
0.0
WT Y949F
NS
WT Y949F
WT Y949F
NS
WT Y949F
WT Y949F
NS
WT Y949F
Control
D12
D12
D12
WT Y949F
WT
Y949F
Control
kDa
225 225
kDa
76 76
kDa
225 225
kDa
225
D18
D18
D18
kDa
225
225
WT Y949F
kDa
76 76
WT Y949F
225
Figure 6 | VEGFA-induced signalling in Vegfr2Y949F/Y949F. (a) VEGFR2 immunoprecipitation (IP) and immunoblotting (IB) for pY1173VEGFR2, VEGFR2,
pY658VE-cadherin and VE-cadherin from lung lysates after tail vein injection of PBS (P) or VEGFA (V) in WTand Vegfr2Y949F/Y949F (Y949F) mice, followed
by circulation for different time periods (P1, P2, P5: PBS injection and circulation for 1, 2 or 5 min; V1, V2, V5, V10, V15, V60: VEGFA injection and circulation for 1, 2, 5, 10, 15 or 60 min). Quantication shown to the right; n 4. Groups of early (V1V2), intermediate (V5, V10, V15) and late (V60) time points were
quantied. Phosphoform/total protein band densities were normalized to the mean of all PBS samples. Two-way ANOVA, upper (p1173VEGFR2): P(genotype) 0.032, P(time) 0.0009; lower (VE-cadherin): P(genotype) o0.0001, P(time) 0.0049. (b) VEGFA-induced accumulation of pY418 Src
(upper) and pT308 Akt (lower) in total lung lysates (TLL) from Vegfr2Y949F/Y949F (Y949F) mice. Quantication as in a, relative to the unphosphorylated
c-Src and Akt pools and normalized to mean PBS values. Two-way ANOVA, upper (Src): P(genotype) 0.0137, P(time) NS; lower (Akt):
P(genotype) NS, P(time) 0.0013. (c) Quantication (top) of IB (bottom) for pY1173VEGFR2 and VEGFR2 in VEGFR2 immunoprecipitates (IP) from
B16F10 tumours at D12 and D18 after inoculation of WT and Vegfr2Y949F/Y949F (Y949F) mice. n 19/WT D12, 14/WT D18, 15/Y949F D12 and 14/Y949F
D18. (d) Quantication (top) of IB (bottom) for pY418Src and c-Src in VEGFR2 immunoprecipitates (IP) from tumour lysates as in c. n 19/WT D12, 13/
WT D18, 14/Y949F D12 and 14/Y949F D18. (e) Quantication (top) of IB (bottom) for VEPTP and VEGFR2 on VEGFR2 immunoprecipitates (IP) from tumour lysates as in c. n 19/WT D12, 14/WT D18, 15/Y949F D12 and 14/Y949F D18 tumours. Data shown as means.e.m. Dashed lines in ce indicate
that data sets from D12 and D18 were not compared statistically. In a,b two-way ANOVA, with Sidaks post hoc test; in ce Students t-test. *Po0.05, **Po0.01, ***Po0.0001, ****Po0.00001. Experiments were performed four independent times.
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WT Y949F
VEGFR2
VE-cad
VEGFR2
VE-cad
VEGF
VEGF
P
TSAd
VEPTP
Src P P
P Src P
Tumour Tumour
Therapy Therapy
Metastasis
Oedema
Inflammatory cells
Inflammatory cells
Molecules
Figure 7 | Schematic outline of Y949 signalling in VEGFA-regulated vascular permeability. WT and Vegfr2Y949F/Y949F (Y949F) endothelial junctions are
shown (upper). In the WT, the junction is active, that is, VE-cadherin is not engaged in homophilic interaction, and VEGFA signalling results in c-Src activation at junctions. In the Y949F mutant, VEGFR2 remains in complex with VEPTP and VE-cadherin, promoting junctional quiescence. c-Src may be activated but not at junctions. Lower schematics shows that junctional activation in the WT results in extravasation of cells and molecules, causing tissue oedema as well as metastatic spread via tumour cell intravasation into the blood stream (cells indicated in green). In the Y949F mutant, extravasation and oedema are blocked and metastatic spread is suppressed, while inammatory cell extravasation is unaffected.
tyrosine phosphorylation of c-Src Y418 increased with VEGFA during the early phase but did not accumulate in the later phase of signalling (Fig. 6b, upper). Thus, the proles of VEGFA-induced, endothelial c-Src phosphorylation differed between the WT and Vegfr2Y949F/Y949F mice. On the other hand, pT308AKT, examined in total lung lysates, accumulated with relatively slow and similar kinetics in the WT and Vegfr2Y949F/Y949F lungs in response to VEGFA injection (Fig. 6b, lower). These data indicate that the VEGFR2-Y949F mutation interfered with c-Src activation and pathways downstream of c-Src, including VE-cadherin phosphorylation at Y658.
VEGFA signalling in B16F10 tumours was examined to understand how leakage was regulated in a tumour context. Immunoblotting of lysates from individual tumours showed that the pY1173/VEGFR2 ratio was signicantly higher at D12 in tumours from WT mice compared with Vegfr2Y949F/Y949F mice (Fig. 6c; lower part shows representative immunoblot), correlating with the VEGFA signalling data from lung lysates (Fig. 6a). At D18, the pVEGFR2/VEGFR2 ratios were similar between the genotypes. The results on the pVEGFR2/VEGFR2 levels are likely to reect the status in the tumour vasculature, as B16F10 cells do not express VEGFR2 (ref. 28). Immunoblotting for pY418Src in the VEGFR2 immunoprecipitates from tumour lysates showed a signicantly higher pSrc/Src ratio in the D12 tumours from WT mice compared with Vegfr2Y949F/Y949F mice (Fig. 6d; lower part shows representative blot). At D18, there was no difference between the genotypes in the extent of pSrc accumulation.
The restriction in VEGFR2 signalling in tumours from the Vegfr2Y949F/Y949F mice at D12 may involve dephosphorylation of VEGFR2 by VEPTP, as we found increased levels of VEGFR2/ VEPTP complexes in the Y949F condition (Fig. 6e; lower part shows representative blot). VEPTP expression levels in D12 tumours did not differ between the genotypes (Supplementary Fig. 5c). At D18, VEGFR2/VEPTP complex formation was similar between WT and Vegfr2Y949F/Y949F-derived tumours (Fig. 6e). For uncropped versions of all blots presented in this study, see Supplementary Figs 613.
We conclude that VEGFA-induced leakage (see Fig. 7 for a schematic outline of VEGFA signalling regulating vascular permeability/leakage) can be restricted during a window of time of B16F10 tumour growth, by suppressing the pY949 pathway. Moreover, the data indicate that VEGFA signalling is dynamically regulated by VEPTP in solid tumours.
DiscussionHere we show that a specic arrest in VEGFA-induced vascular permeability leading to attenuation of molecular extravasation in organs such as the trachea and skin is compatible with normal vascular development, morphology and function. The Vegfr2Y949F/Y949F mouse displayed reduced vascular leakage in several orthotopic tumour models and a signicant reduction in metastatic spread of insulinoma and melanoma (see Fig. 7 for a schematic summary of the results). The enhanced vascular barrier in the Vegfr2Y949F/Y949F mouse led to reduced tumour oedema,
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11017
allowing for increased chemotherapeutic efcacy. While our data strongly indicate that signalling via pY949-VEGFR2 regulates c-Src activation and VE-cadherin phosphorylation and therefore adherens junction integrity, we do not rule out the involvement of transendothelial pores/vesiculovacuolar organelles in VEGFA-induced vascular permeability29.
Induced vascular permeability, also denoted vascular hyperpermeability or vascular leakage, may be acute or chronic30. The best studied factors regulating vascular leakage include VEGFA and inammatory cytokines such as histamine and bradykinin31. An implication from the current study is that VEGFA dominates over inammatory cytokines in regulating molecular extravasation, in the models studied here. VEGFA was originally identied as a vascular permeability factor30, but the physiological role of vascular permeability regulated by VEGFA/ VPF has remained unclear. Dvorak and colleagues implicated vascular permeability in the formation of a provisional matrix for the growth of new vessels32. Our data do not support an essential role for the VEGFA-driven pathway regulated by VEGFR2 pY949 signalling, in angiogenesis.
Cheresh and coworkers have shown the critical role for c-Src-pathways in regulation of VEGFA-induced vascular permeability25. Our work extends these data by implicating pY949-VEGFR2/TSAd in regulation of c-Src pY418, VE-cadherin pY658 and the stability of adherens junctions. VE-cadherin is constitutively tyrosine phosphorylated in postcapillary venules compared with arteries/arterioles and regulated through ow-induced activation of c-Src, as judged from immunouorescent staining of different capillary beds27. In this study, we noted a marked reduction in VE-cadherin Y658 phosphorylation in the Vegfr2Y949F/Y949F lung lysates compared with WT, in agreement with the altered c-Src phosphorylation prole (Fig. 6a,b) and with the persistence of adherens junctions in response to VEGFA, in vivo and in vitro (Fig. 1gj). However, the complex biology of c-Src precludes a direct causeeffect relationship between c-Src pY418 phosphorylation and VE-cadherin/adherens junction stability. We have previously shown that pY418 Src accumulates with relatively slow kinetics in vivo and that it translocates to junctions in complex with the adaptor molecule TSAd8. It is possible that the dynamics of c-Src activation differs between different subcellular compartments such as junctions and focal adhesions. Thus, at focal adhesions, c-Src activity is induced as a consequence of cell adhesion-dependent focal adhesion kinase activation, which also affects vascular permeability most likely by orchestrating cell shape33. In agreement with the data presented here, focal adhesion kinase (Ptk2) gene knockout decreases tumour spread34. Owing to technical constraints imposed by the quality of commercial c-Src antibodies, we have not been able to distinguish different subcellular pY418 Src pools in vivo. However, accumulation of pY418 Src in relation to the total c-Src pool was decreased both in Vegfr2Y949F/Y949F lung endothelial cells and in B16F10 tumours growing in mutant mice.
VEGFA-induced accumulation of pT308 Akt was similar in the WT and mutant Y949F VEGFR2 conditions. Akt plays an important role in vascular permeability induced by inammatory cytokines26. Akt is critical for the activation of endothelial NO synthase (eNOS) in vivo, by phosphorylation on Ser1176 (ref. 35), resulting in increased NO production. NO, a potent regulator of the vascular tone, mediates vasodilation by stimulating soluble guanylyl cyclase and increasing cyclic GMP in smooth muscle cells36. In this study, we recorded transiently reduced blood pressure indicative of vasodilation in response to VEGFA in the descending aorta (Fig. 2j) in both WT and Vegfr2Y949F/Y949F mice. This result together with the full activation of Akt, judged from the pattern of pT308 Akt accumulation in the mutant, indicate
that the NO pathway is intact in the Vegfr2Y949F/Y949F mouse. eNOS has previously been implicated in tumour vascular permeability, and inhibition of eNOS activity by a small caveolin-derived peptide (cavtratin) leads to suppressed tumour vascular permeability and reduced primary tumour growth without affecting blood ow and angiogenesis37. In contrast, while metastatic spread was suppressed in the Vegfr2Y949F/Y949F mouse, primary tumour growth was only modestly affected.
We noted reduced phosphorylation of VEGFR2 at phosphosite Y1173 in the Vegfr2Y949F/Y949F lungs and in D12 B16F10 melanoma. Mutation of the human tyrosine residue, Y951 (corresponding to mouse Y949), in a puried recombinant intracellular VEGFR2 domain interferes with protein folding38. Whether protein folding is affected also in the full-length VEGFR2 remains to be shown. We noted that the level of phosphorylation of the full-length mouse VEGFR2-Y949F mutant relative to WT VEGFR2 varied during tumour progression. Thus, in B16F10 tumours, the relative level of pY1173 in WT VEGFR2 was higher than for VEGFR2-Y949F at an early stage, whereas the levels were similar at later stages of tumour growth. We propose that the reduced VEGFR2 phosphorylation in the Vegfr2Y949F/Y949F lungs in D12 B16F10 tumours at least in part was a consequence of sustained VEGFR2/VEPTP complex formation. VEPTP is a receptor-type phosphotyrosine phosphatase specically expressed in endothelial cells and known to have VE-cadherin and Tie2 as direct substrates39. VEGFR2 is an indirect substrate for VEPTP, resulting in dephosphorylation of VEGFR2 in complex with Tie2 (ref. 24). Here we conrm and extend the data on the important role of VEPTP as an in vivo regulator of VEGFR2 activity. VEGFR2/VEPTP complex formation correlated with VEGFR2 pY1173 levels and the extent of oedema. Thus, B16F10 melanomas in WT mice showed low VEGFR2/VEPTP complex formation, high p1173 VEGFR2 levels and prominent oedema. In contrast, melanomas in the Vegfr2Y949F/Y949F mice displayed high VEGFR2/VEPTP complex formation, low p1173 pVEGFR2 levels and reduced oedema. With time, VEPTP/VEGFR2 complex formation decreased in tumours from both WT and Vegfr2Y949F/Y949F mice. Still, the VEGFR2 and c-Src activities were suppressed in the late stage of tumour growth in WT mice, showing a restriction in VEGFA signalling independent of VEPTP. The gradual progression towards lower VEGFR2 phosphotyrosine levels over time is in agreement with models for resistance towards anti-VEGFA neutralizing therapy in cancer40.
The extent of tumour oedema correlates to the blood supply, the volume of blood entering the tumour and the lymphatic drainage41. Tumour blood supply decreases as the tumour mass increases42, most likely due to a progressive deterioration of the tumour vasculature with eventually fewer functional vessels. Tumours in Vegfr2Y949F/Y949F mice were less oedematous, which we attribute to the higher vessel integrity in the mutant. Excess and persistent extravasation increases the interstitial pressure and impairs delivery of therapeutics to the tumour tissue4. In agreement, the B16F10 tumour-bearing Vegfr2Y949F/Y949F mice were more responsive to treatment with chemotherapy. Complete sealing of junctions to VEGFA, as well as to inammatory cytokines, as a result of expression of a VE-cadherin/a-catenin fusion protein, leads to a partial reduction in inammatory cell extravasation43. In contrast, restricting VEGFA-induced permeability alone as in the Vegfr2Y949F/Y949F genotype, failed to suppress inammatory cell inltration in RIP1-TAg2 insulinomas (Supplementary Fig. 3ce) and B16F10 melanomas (Supplementary Fig. 3hj). While we do not exclude that acute inammation or other aspects of inammation not studied here are affected by the VEGFR2 mutation, our data suggest that there is no direct relationship between the capacity to spread metastasis
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and primary tumour inammation. Moreover, lodging of circulating tumour cells was unaffected by the mutation (Supplementary Fig. 4b) suggesting that the stable Vegfr2Y949F/
Y949F junctions may restrict the ability of tumour cells to enter but not exit the circulation. The caveats of the experimental metastasis model44 (that is, injection of tumour cells in the tail vein followed by lodging of tumour cells in the lung) compared with spontaneous metastasis, limits conclusions however.
Possibly, factor(s) other than VEGFA may contribute to extravasation of B16F10 cells in the lung; indeed, the Vegfr2Y949F/Y949F endothelium retains sensitivity to other permeability-inducing factors than VEGFA such as histamine. It is pertinent to ask whether the reduced metastatic spread in the Vegfr2Y949F/Y949F tumours can be attributed directly to the VEGFA-resistant Y949F junctions, that is, do tumour cells intravasate into the circulation via junctions? Alternatively, were other parameters, established as a consequence of the nonleaky junctions and the reduced oedema, more important? As we did not record signicant changes compatible with vascular normalization4, that is, in vascular density, pericyte coating, necrosis or inammation in the B16F10 melanomas from WT and mutant mice, we propose that the Vegfr2Y949F/Y949F phenotype primarily depends on the VEGFA-resistant junctions.
In conclusion, our data suggest that specically suppressing VEGFA-induced vascular permeability without attenuation of vital aspects of VEGFA biology, for example, endothelial survival and neoangiogenesis, may offer an attractive strategy in future drug development to reduce oedema, to improve therapeutic efcacy and to suppress metastatic spread in cancer.
Methods
Vegfr2Y949F/Y949F mouse model. A mouse model with knock-in of phenylalanine(F) to replace the tyrosine (Y) at position 949 (Y951 in the human) of VEGFR2 (designated Vegfr2Y949F/Y949F) in a mixed 129S6/C57BL/6 was created using VelociGene technology (Regeneron Pharmaceuticals, New York, USA)11. The Vegfr2Y949F/Y949F mouse was backcrossed onto the C57BL/6 background for more than 10 generations and maintained through crossing of Vegfr2Y949F/Y949F heterozygotes. All data shown are based on comparison of Vegfr2Y949F/Y949F and WT littermates obtained from heterozygote crossings in experiments repeated at least three times. The Uppsala University board of animal experimentation approved all animal work; efforts were done to minimize suffering as supervised by the University veterinarian. Mice were anaesthetized with ketamine/xylazine, avertin or isourane (Isoba, 3% induction and 1.5% maintenance).
Statistical analysis. For parametric comparison, Students t-test was performed to compare means between two groups. One-way ANOVAs were performed to compare 42 treatment groups and two-way ANOVA was used when two factors were involved, for example, treatment and genotype. Repeated measures ANOVA was used for tumour growth curves. Multiple comparisons post hoc tests were chosen based on how many group comparisons were made. When all groups were compared with each other, Tukeys Honestly Signicant Difference (HSD) test was used, while Sidaks test was used when individual time points were compared with each other. Non-parametric MannWhitney U-test/Wilcoxon sign-rank test (for paired comparison) was used to compare two groups, while KruskalWallis test followed by Dunns multiple comparisons was used for 42 group-wise comparisons. GraphPad Prism 6.0 was used for statistical analyses. The threshold for statistical signicance was set at a 0.05 (*Po0.05, **Po0.01 and ***Po0.0001).
Antibodies and growth factors. All antibodies used in this study, their origin and working concentrations are listed in Supplementary Table 1.
Recombinant mouse VEGFA164 was purchased from Peprotech EC Ltd (Rocky Hill, NJ, USA) and used for in vitro analyses. Dog VEGFA164, which is 99% identical to mouse VEGFA, was used for in vivo analyses and was produced at the Paul-Scherrer Institute, Villigen, Switzerland. In vitro tests showed no difference in VEGFR2-stimulating activity of commercial mouse VEGF and in-house-produced dog VEGF. Histamine was from Sigma-Aldrich.
Orthotopic mouse glioma model. Mice (homozygous Vegfr2Y949F/Y949F and WT littermates) were inoculated with GL261 glioblastoma cells (a kind gift from Geza Safrany, NRIRR, Budapest). Cells were not veried but tested negative for mycoplasma using the Mycoplasma detection kit (Lonza). GL261 is a chemically induced murine cell line, which when inoculated orthotopically has many features
reminiscent to human glioma18. Cells were cultured in DMEM Glutamax medium containing 10% FCS, washed and resuspended in PBS for inoculation. Mice were anaesthetized with isourane (Isoba, 3% induction and 1.5% maintenance). A small incision was made rostrocaudally along the midline of the scalp. A small hole was drilled at stereotaxic coordinates 1.1 mm anterior and 2.1 mm lateral to bregma.
A volume of 0.5 ml of cells (1.5 105 cells per ml) were slowly injected using a
Hamilton microsyringe with a 33 G needle at 2.9 mm depth below the dura. The needle was left in place for 3 min before slow retraction over 2 min. The wound was closed using 6-0 resorbable suture, and analgesia carprofen (Rimadyl Vet, Orion Pharma, Sweden; 50 mg per 20 g mouse) was injected subcutaneously on the back.
Tumours were left to grow for 18 days. On the last day, mice were intravenously injected with 150 mg per 20 g mouse of Alexa-555-Cadaverine (Life Technologies)
together with 100 mg of biotinylated tomato lectin in PBS. After 15 min of circulation, mice were killed using an overdose of ketamine/xylazine (see above) and transcardially perfused with physiological saline followed by cold 4% paraformaldehyde (PFA). Brains were postxed for 4 h and transferred to 25% sucrose for cryoprotection. Brains were coronally cut on a vibratome at 80 mm thickness and mounted on cover glass in series to cover the entire volume of the tumour. Tumour volume was estimated using haematoxylin-and-eosin-stained sections throughout the brain. Representative sections were immunostained for lectin (Streptavidine-Alexa-488) and nuclei (Hoechst). The two largest (mid) sections of each tumour were used for quantication of vessel density and Cadaverine-555 accumulation in the brain, indicating abnormal leakage.
RipTag spontaneous endocrine tumour and metastasis model. Vegfr2Y949F/Y949F mice were crossed with RIP-Tag2 mice on the C57Bl/6 background. Vegfr2Y949F/Y949F heterozygous offspring was bred to obtain RIP-Tag2:WT and RIP-Tag2:Vegfr2Y949F/Y949F homozygous mice. All analyses were done using mice of mixed gender aged 14 weeks. Islets larger than 1 1 mm were dened as Riptag
tumours, and overtly red islets o1x1 mm were dened as angiogenic islets as assessed under a stereological microscope. Tumours and pancreas including angiogenic islets were sectioned and stained to analyse vessel characteristics. For quantication of metastases, right liver lobes were sectioned in their entirety. In all, 30 consecutive sections were examined for score of metastases based on a cluster of at least 25 cells, which were conrmed by SV40-Tag staining. Average numbers of liver metastases per section was calculated.
B16F10 subcutaneous primary tumour and metastasis model. B16F10 cells (American Type Culture Collection) were not validated after purchase but retained melanin production and were regularly tested negative for mycoplasma using the Mycoplasma Detection Kit (Lonza). Cells (0.5 106 per 100 ml) were injected sub
cutaneously in the mouse back skin. Tumour size was measured with a caliper every other day from D6 and tumours were collected at D12 or D18 after implantation. Tumour wet and dry weights were measured to determine water content/oedema in the tumours, calculated (tumour wet weight tumour dry weight)/tumour wet weight
and given as percentage (%). Tumour vessel characteristics were analysed after injection of FITC-Lectin (Sigma) in the tail vein, followed by 20-min circulation and then, a washout with PBS via the left cardiac ventricle. Snap-frozen tumours were sectioned, stained and imaged using a Zeiss Axioimager. Quantication of vessel parameters was done using ImageJ (National Institutes of Health) in a blinded fashion. To score for metastatic spread, lentivirus-transduced DsRed-expressing B16F10 cells (kindly provided by Professor David D. Schlaepfer Department of Pathology, La Jolla, CA) were injected in the mouse right ear dermis (4 104 cells in
10 ml growth factor-depleted Matrigel; BD). Tumour volume was measured with a caliper every other day. At D15, mice were perfused with PBS via the left cardiac ventricle and lungs were removed, xed and frozen. For quantication of metastases, the left large lung lobe from each mouse was sectioned in its entirety. Lung metastases were enumerated by counting DsRed-positive lesions from confocal microscopy in four sections spaced 100 mm apart, in each specimen.
Microsphere extravasation in mouse trachea and Riptag tumours. Twenty microlitres of 30 nm Fluoro-Max Green Aqueous Fluorescent microspheres (Thermo Scientic, Fremont, CA, USA) together with 80 ml solution of VEGFA164 (5 mg per 20 g body weight) or PBS were administered by tail vein injection. Mice were transcardially perfused using 1% PFA in PBS 1 h later. For histamine-induced microsphere extravasation, 20 ml of 30 nm uorescent microspheres was injected together with histamine solution (80 ml; 1.25 mg per 20 g body weight). Perfusion with 1% PFA was performed after 2 min circulation45. Tracheas were dissected and xed in 1% PFA in PBS for an additional 2 h, stained with endothelial cell markers to visualize the vasculature. To observe microsphere extravasation in Riptag tumours, 100 ml of 30-nm uorescent microspheres was administered by tail vein injection. Intravascular microspheres were removed by PBS perfusion via the left ventricle 1 h later. Riptag tumours were postxed in 2% PFA for 6 h and kept in 30% sucrose overnight at 4 C followed by embedding in cryosectioning medium. Tumours were sectioned and stained for endothelial cell markers. Imaging was performed using a Zeiss700 confocal microscope or a Zeiss 710 Structured Illumination microscope.
TMZ chemotherapy. B16F10 cells (0.5 106 per 100 ml) were injected
subcutaneously in the mouse back skin. From D4 to D8, TMZ dissolved in
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dimethylsulfoxide to a nal concentration of 2%, was administered i.p. every day at 10 mg kg 1, a dose established to have no effect on B16F10 growth in WTmice from test studies. Control mice were treated with a similar volume of 2%
dimethylsulfoxide. Tumour size was measured with a caliper every other day from D6 until D18 after implantation.
DsRed-expressing B16F10 cell lung colonization. DsRed-expressing B16F10 cells (1 million in 100 ml PBS) were injected into mouse tail vein. At 48 h, mice were perfused with PBS via the left cardiac ventricle, and lungs were removed, xed and frozen. Left lung lobes were sectioned in their entirety. Tumour cell foci were enumerated by counting DsRed-positive lesions in confocal microscopy in two sections spaced 400 mm apart and ve elds per section.
Miles assay. Mice (females, 68 weeks) were subjected to i.p. injection with pyrilamine maleate salt (4 mg per kg body weight in 0.9% saline, Sigma) to inhibit unspecic histamine release (for example, by pulling the skin), 30 min before Evans blue tail vein injection (100 ml 1% Evans blue in sterile saline, Sigma).
This was followed by intradermal injection of VEGF (100 ng in 50 ml per site) or sterile saline 10 min after Evans blue administration. Thirty minutes after the VEGF injection, the dorsal skin was excised and Evans blue was extracted by immersion in formamide buffer. Absorbance at 620 nm was measured using a spectrophotometer (EMax microplate reader, Molecular Devices, CA, US). Extravasated Evans blue was determined from a standard curve and normalized to tissue weight (g).
Isolation of lung endothelial cells. Mouse lungs were collected at P10 or34 weeks, minced and digested in 10 ml Dulbeccos PBS medium containing 2 mg ml 1 collagenase type I (Sigma), for 1 h at 37 C with shaking, followedby ltration through a 70-mm disposable cell strainer (BD Falcon). Cells were centrifuged at 400g for 8 min at 4 C, suspended in cold PBS with 0.1% bovine serum albumin (BSA) and incubated with anti-rat immunoglobulin G-coated magnetic beads (Dynabeads sheep anti-Rat IgG, Invitrogen) pre-coupled with rat anti-mouse platelet/endothelial cell adhesion molecule-1 (PECAM-1; MEC13.3, BD Pharmingen, 553370) for 10 min, with gentle agitation. Beads were separated from the solution with a magnetic particle concentrator (Dynal MPC-S, Invitrogen). The beads were washed ve times with PBS, and endothelial cells were suspended in MV2 growth medium with the following supplements: 5 ng ml 1 endothelial growth factor; 0.2 mg ml 1 hydrocortisone; 0.5 ng ml 1 VEGFA; 10 ng ml 1 basic broblast growth factor; 20 ng ml 1 insulin-like growth factor-1 (Promocell); and 5% penicillin/streptomycin (Sigma). The purity of cells was 495% as determined by staining for VE-cadherin. The isolated lung endothelial cells were cultured on slides pre-coated with gelatin. Cells were treated with or without 100 ng ml 1 mouse VEGFA164 for 10 min before xation in 4% PFA in PBS for 10 min, permeabilized in 0.1% Triton X-100 in PBS for 30 min, followed by immunouorescent staining. Images were acquired using a Zeiss LSM700 confocal microscope ( 63, numerical aperture 1.4 objectives).
Immunoprecipitation and immunoblotting. For in vitro signal transduction analyses, endothelial cells were isolated from lungs of P10 pups as described above, and cultured. Cells were treated with or without 100 ng ml 1 mouse VEGFA164 (PeproTech) for 8 min and lysed for subsequent immunoprecipitation and immunoblotting.
For in vivo signal transduction analyses, lung lysates were used from mice at 1, 2, 5, 10, 15 or 60 min after injection of dog VEGFA164 in the tail vein. An equal volume of PBS was injected into control mice. B16F10 tumour lysates were obtained from mice at D12 or 18 after B16F10 cell implantation. Tissues were lysed in commercial RIPA buffer (ProteinSimple) followed by immunoprecipitationfor VEGFR2 and immunoblotting, or immunoblotting of total lysates. For immunoprecipitation, 1.5 mg/ml tissue lysate were diluted in RIPA buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP 40) supplemented with protease inhibitors (Roche, 04693116001), phosphatase inhibitors (Thermo scientic, 88667), 1 mM NaF, 1 mM Na3VO4. Lysates were precleared by incubating with 25 ml of Protein
G Sepharose 4 Fast Flow beads (Ge Healthcare, 17-0618-01) per sample for 2 h at 4 C. Precleared lysates were incubated with 1.5 mg VEGFR2 antibody for 2 h at4 C and 25 ml of Protein G beads were added and incubation continued for 1 h (lung lysates) and overnight (B16 tumor lysate and isolated lung endothelial cells). Sepharose beads were washed 5 times with 1 ml RIPA buffer per sample without protease and phosphatase inhibitors and collected by centrifugation at 3000Xg for 1 min at 4 C. 30 ml of 2X LDS sample buffer were added to each sample and heat at 70 C for 10 min. Immunocomplexes were resolved on NuPAGE Novex 412% Bis-Tris gels (Invitrogen) and processed for immunoblotting8. Full scans of all immunoblots are presented in Supplementary Figs 613.
Immunouorescent staining. Eyes were removed and prexed in 4% PFA for 20 min at room temperature. After dissection, retinas were blocked overnight at4 C in 1% FBS (Gibco), 3% BSA (Sigma) and 0.5% Triton X-100 (Sigma). Retina samples were incubated over night with primary antibodies in blocking reagent, followed by washing and incubation with Isolectin-B4 in Pblec (1 mM MgCl2,
1 mM CaCl2, 0.1 mM MnCl2 and 1% Triton X-100 in PBS) overnight. Thereafter,
samples were incubated with the appropriate secondary antibody for 2 h at room temperature, and mounted in uorescent mounting medium (DAKO). Images were acquired using a Zeiss700 confocal microscope. For analysis of the activation state of endothelial junctions in the retinal vasculature, the unbiased software (Matlab)15 was used.
Blood parameter measurements. The blood ow measurements were performed with a microsphere technique46 as described below: WT and Vegfr2Y949F/Y949F mice 34 months old were anaesthetized with isourane and placed on an operating table, where the animals body temperature was maintained constant at 38 C. A polyethylene tracheal tube was inserted to ensure free airways and polyethylene catheters were inserted into the ascending aorta, via the right carotid artery, and into the right femoral artery. The former catheter was connected to a pressure transducer (Physiologic Pressure Transducer; AD Instruments, Oxford, UK) to allow continuous monitoring of the mean arterial blood pressure, which was allowed to stabilize for 2025 min. Approximately 105 microspheres (EZ-Trac; Triton Technology, San Diego, CA, USA) with a diameter of 10 mm were injected for 10 s via the catheter with its tip in the ascending aorta. Starting 5 s before the microsphere injection, and continuing for a total of 60 s, an arterial blood reference sample was collected by free ow from the catheter in the femoral artery at a rate of0.10 ml min 1. The exact withdrawal rate in each experiment was conrmed by weighing the sample.
Arterial blood was then collected from the catheter in the femoral artery and analysed for blood pH, pCO2, pO2, oxygen saturation, S-HCO3 , base excess,
S-Na , S-K , haematocrit and haemoglobin concentration, with an iStat (Abbott Scandinavia). The cassette used for this analysis requires 90 ml of whole blood. Arterial blood was also used for the determination of blood glucose concentrations (Medisense Test Reagent Strips; MediSense) and for serum insulin determinations with ELISA (Rat Insulin ELISA; Mercoedia AB).
The animals were killed, and the organs were carefully dissected free from fat, blotted and weighed. The microspheres in the organs and the arterial blood reference sample were visualized by a freeze-thawing technique and counted by an observer blinded for the origin of the samples47.
The blood ow values were calculated according to the formulaQorg Qref Norg/Nref, where Qorg is organ blood ow (ml min 1), Qref is
withdrawal rate of the reference sample (ml min 1), Norg is the number of microspheres present in the organ and Nref is the number of microspheres present in the reference sample46.
Blood pressure measurements after VEGF administration. Mice (females, B15 weeks) were anaesthetized by an intraperitoneal (i.p.) injection of avertin48. A polyethylene tracheal tube was inserted to ensure free airways, and a polyethylene catheter was inserted into the descending aorta, via the right femoral artery, and connected to a pressure transducer (AD Instruments) connected to a PowerLab system (AD Instruments. When the blood pressure had remained stable for 10 min an injection of 0.1 ml of saline or VEGFA (5 mg per 20 g body weight) was given in the right jugular vein. Mean arterial blood pressure was then registered for 15 min.
E11.5 Hindbrain vascularization. Embryos at E11.5 were collected and xed in 4% PFA overnight. Hindbrains were dissected the following day and thereafter, brains were permeabilized in a buffer containing 1% Triton X, 0.01 mM CaCl2, 0.1 mM
MgCl2, 0.1 mM MnCl2, in PBS (pH 6.8), for two 15-min washes and subsequently incubated with biotinylated isolectin-B4 (L-2140, Sigma-Aldrich, Sweden).
A streptavidine-coupled Alexa-488 (Life Technologies) was used for visualization. Hindbrains were at-mounted and imaged in a confocal (LSM700). The image analysis was done using CellProler (http://www.cellprofiler.org
Web End =www.cellproler.org) with a pipeline as follows: an initial segmentation of the vessels was done using a xed global threshold, followed by ltering out small particles. Next, a skeleton was computed from the binary vessel mask. From the skeleton, the number of branch points and the branch lengths were measured. Branches touching the border or very short branches were removed. For each branch, the mean distance values for the corresponding skeleton segment were calculated.
Transmission electron microscopy. Two sex- and age-matched mice (7-week-old male and female Vegfr2Y949F/Y949F and WT littermates) were perfusion xedand their organs were collected and embedded in epoxy resin16. Briey, mice were anaesthetized using a mixture of ketamine/xylazine and perfused via the left heart ventricle with oxygenated DMEM/1 mM CaCl2/5 mM D-glucose. This was immediately followed by perfusion with 3% PFA/1.5% glutaraldehyde/5% sucrose in 0.1 M phosphate buffer, pH 7.2. Organs (kidney and pancreas) were removed, cut in small blocks and further xed (1 h at room temperature) in2% glutaraldehyde in 0.1 M sodium cacodylate buffer, rinsed in 0.1 M cacodylate, postxed (1 h, on ice, in the dark) in 1% OsO4, stained (over night, room temperature, in the dark) with 2% uranyl acetate, dehydrated in graded ethanol and LX-112 embedded (Ladd Research, Burlington, VT). Ultrathin sections (B30 nm)
were obtained using an oscillating diamond knife (Diatome, Emsdiasum), further stained with 2% uranyl acetate followed by lead citrate, examined and imaged with a Jeol 1010 electron microscope using a bottom-mount AMD camera. Images were exported in TIFF format, and labelled and assembled using Adobe Photoshop and
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Illustrator CS6 (Adobe Systems). In some images, the contrast and brightness were post-processed in Adobe Photoshop using the respective linear functions.
RNA extraction and sequencing. RNA was extracted using the Qiagen RNeasy kit following the manufacturers instructions and using lungs from 5- to 8-week-old mice. Extracted RNA quality was assessed with a BioAnalyzer (Agilent Technologies, Wilmington, DE, USA) and concentration estimated using the Qubit RNA BR Assay Kit (Thermo Ficher Scientic, Eugene, Oregon, USA). From 11 mg of total RNA, mRNA purication was performed using Dynabeads mRNA Purication Kit (Ambion, Life Technologies AS, Oslo). mRNA libraries, three biological replicates per sample, were prepared using the Ion Total RNA-Seq Kit v2 (Life Technologies) using the standard protocol, and sequencing was performed on Ion Proton sequencer (Life Technologies). Libraries were barcoded three per PI v2 chip to achieve 20 million reads per replicate. Reads were processed, mapped to a reference genome (mm10) and summarized using Torrent Suite version 5.0.2 and default settings. The statistical analysis of gene expression was performed in R using DESeq2 package (http://dx.doi.org/doi:10.1186/gb-2010-11-10-r106
Web End =doi:10.1186/gb-2010-11-10-r106) with dispersion t type set to local. The FPKM (fragments per kilobase per million) function from DESeq2 package was used to normalize reads per gene as RPKM (reads per kilobase per million), using the robust library size estimate. The RPKM values for selected genes were plotted on the log10 scale based on the folds of our genes of interest established in log2 fold change (log2FC 1 means the gene is upregulated twofolds).
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Acknowledgements
This work was supported by grants from the Swedish Cancer Society, the Swedish Science Council, the Knut and Alice Wallenberg Foundation and the Association for International Cancer Research/Worldwide Cancer Research (grant 13-1295), to L.C.-W., from the Swiss National Science Foundation (grant 31003A-130463, the Oncosuisse (grant OC20120008 2007) to K.B.-H., from the European Research Council (294556 BBBARRIER) and Leducq Foundation (Sphingonet) to C.B., and from the National Institute of Health (CA172983 and CA175592) to R.V.S. E.G. was supported by a Wenner-Gren Foundation postdoctoral scholarship. We acknowledge expert assistance by the following SciLifeLab facilities: NGI; BioVis imaging facility; BILS; and UPPMAX/ SNIC. We also thank Petter Ranefall, Center for Image Analysis, SciLifeLab, Uppsala
NATURE COMMUNICATIONS | 7:11017 | DOI: 10.1038/ncomms11017 | http://www.nature.com/naturecommunications
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11017
University, for expert assistance with image rendering and analysis. We thank Dr Doug Hanahan, Swiss Institute for Experimental Cancer Research, for antibodies against SV40 T, Dr Gza Sfrny, Frdric Joliot-Curie National Research Institute for Radiobiology and Radiohygiene, Budapest, Hungary, for sharing the GL261 cells and Dr David D. Schlaepfer, University of California at San Diego for dsRed-B16F10 cells.
Author contributions
X.L., N.P., E.O.S., N.H., F.R., M.S.-J., E.G., K.B., C.T., R.V.S., V.T., L.J. and L.C.-W. planned and executed experiments; all authors analysed and interpreted data, and assisted in putting the manuscript together.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications
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Competing nancial interests: The authors declare no competing nancial interests.
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How to cite this article: Li, X. et al. VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread. Nat. Commun. 7:11017 doi: 10.1038/ncomms11017 (2016).
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Copyright Nature Publishing Group Mar 2016
Abstract
The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2Y949F/Y949F leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2Y949F/Y949F mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2Y949F/Y949F mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms.
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