In Vivo FRET Imaging of Tumor Endothelial Cells Highlights a Role of Low PKA Activity in Vascular Hyperpermeability

Home , Guanine, IBMX

Published OnlineFirst August 3, 2016; DOI: 10.1158/0008-5472.CAN-15-3534 Cancer Microenvironment and Immunology Research

In Vivo FRET Imaging of Tumor Endothelial Cells Highlights a Role of Low PKA Activity in Vascular Hyperpermeability Fumio Yamauchi1,2, Yuji Kamioka1,3, Tetsuya Yano2, and Michiyuki Matsuda1

Abstract

Vascular hyperpermeability is a pathological hallmark of receptor is a canonical inducer of vascular hyperpermeability cancer. Previous in vitro studies have elucidated roles of various and a molecular target of anticancer drugs, we examined the signaling molecules in vascular hyperpermeability; however, causality between VEGF receptor activity and the PKA activity. the activities of such signaling molecules have not been exam- Motesanib, a kinase inhibitor for VEGF receptor, activated ined in live tumor tissues for technical reasons. Here, by tumor endothelial PKA and reduced the vascular permeability in vivo two-photon excitation microscopy with transgenic mice in the tumor. Conversely, subcutaneous injection of VEGF expressing biosensors based on Forster€ resonance energy trans- decreased endothelial PKA activity and induced hyperperme- fer, we examined the activity of protein kinase A (PKA), which ability of subcutaneous blood vessels. Notably, in cultured maintains endothelial barrier function. The level of PKA activity human umbilical vascular endothelial cells, VEGF activated was signiﬁcantly lower in the intratumoral endothelial cells PKA rather than decreasing its activity, highlighting the remark- than the subcutaneous endothelial cells. PKA activation with a able difference between its actions in vitro and in vivo.Thesedata cAMP analogue alleviated the tumor vascular hyperpermeabil- suggested that the VEGF receptor signaling pathway increases ity, suggesting that the low PKA activity in the endothelial vascular permeability, at least in part, by reducing endothelial cells may be responsible for the tumor-tissue hyperpermeabil- PKA activity in the live tumor tissue. Cancer Res; 76(18); 1–11. ity. Because the vascular endothelial growth factor (VEGF) 2016 AACR.

Introduction the intercellular gaps of endothelial cells (15). The inhibition of VEGF and VEGF receptors (VEGFR) results in a decrease of Blood vessels in tumors are signiﬁcantly different from those in permeability and the normalization of tumor vasculature, which normal tissues in terms of structure and function (1). For example, in turn results in the inhibition of tumor growth and improve- vascular permeability is markedly increased in tumor tissues, and ment of anticancer chemotherapy (7). this increase is associated with angiogenesis and metastasis (2–6). The VEGF-induced increase in vascular permeability is medi- Accordingly, suppression of the vascular permeability results in ated by various intracellular signaling pathways, including those the inhibition of tumor growth and metastasis (7). of PTPs, Src, PI3K, uPA, PLC-g, and eNOS (6). Another signaling The high vascular permeability in tumor tissues is caused by molecule that has been shown to regulate vascular permeability various factors, including the structural abnormality of blood is cAMP. In microvascular endothelial cells, cAMP mediates vessels (2, 8), pressure or concentration gradients between com- endothelial barrier function by suppressing vascular permeability partments, and the properties of endothelial cells and/or pericytes (16, 17). Further studies have shown that increased cAMP triggers (2, 6). Among a number of molecules that regulate blood vessels sequential activation of protein kinase A (PKA), a guanine nucle- in tumor tissues, vascular endothelial growth factor (VEGF) has otide exchange factor Tiam1, and a small GTPase Rac1, resulting been a focus of intensive research (6, 9). VEGF increases vascular in an increase in barrier function (18, 19). On the other hand, permeability by promoting transcytosis (10–14) and enlarging cAMP can also contribute to the endothelial barrier function via cAMP-dependent guanine nucleotide exchange factor Epac/ cAMP-GEF and a small GTPase Rap1 (20, 21). Notably, the 1Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 2Medical Imaging System contribution of these potential effectors downstream of VEGFR Development Center, R&D Headquarters, Canon Inc., Japan. 3Innova- to the hyperpermeability in tumor tissue has not been assessed in tive Techno-Hubfor Integrated Medical Bio-Imaging, Kyoto University, live tissues. Kyoto, Japan. Visualization of tumor tissues by two-photon excitation Note: Supplementary data for this article are available at Cancer Research microscopy has opened new windows into the dynamics of Online (http://cancerres.aacrjournals.org/). angiogenesis and the involvement of hematopoietic cells in Corresponding Author: Michiyuki Matsuda, Department of Pathology and tumorigenesis (22, 23). Meanwhile, using cancer cells expressing Biology of Diseases, Graduate School of Medicine, Kyoto University, Yoshida- Forster€ resonance energy transfer (FRET) biosensors, the effects of Konoe-Cho, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-4421; Fax: 81- anticancer drugs on the target molecules have been visualized in 75-753-4655; E-mail: [email protected] the xenograft/homograft recipient mice, revealing considerable doi: 10.1158/0008-5472.CAN-15-3534 heterogeneity among cancer cells (24, 25). To conduct similar 2016 American Association for Cancer Research. approaches for the host cells, transgenic mice expressing the FRET

www.aacrjournals.org OF1

Yamauchi et al.

biosensors are needed. We and others have developed such MetaMorph software (Molecular Devices) as described previously transgenic mice, collectively designated FRET mice, for the visu- (26, 31). alization of activities of protein kinases and small GTPases in vivo (26, 27). Here, by FRET imaging of the intratumoral blood In vivo observation of the vascular endothelial cells vessels, we show that the basal activity of tumor endothelial PKA is Mice were anesthetized with 1.5% to 2% isoflurane inhala- fi signi cantly lower than that of normal endothelial cells and that tion and placed in the prone position. Skin flap was then placed the cAMP analogue could alleviate the increased vascular perme- on a cover-glass. As controls, subcutaneous capillaries with a ability in the tumors. diameter of less than 15 mm and subcutaneous arterioles or venules from 15 to 50 mm in diameter were also observed in Materials and Methods each experiment. Images were acquired every 2 or 4 minutes at Cells and reagents a scan speed of 2 ms/pixel. During imaging, reagents were Panc-02 mouse pancreatic cancer cells were obtained from administered in 100 mL PBS via the orbital plexus, if necessary: the NIH (Bethesda, MD). B16-F10 mouse melanoma cells Motesanib (50 mg/kg), VEGF164 (5 mg/mouse), dbcAMP were obtained from the ATCC. Primary human umbilical (50 mg/kg), 6-Bnz (50 mg/kg), 007 (50 mg/kg), and Qtracker vascular endothelial cells (HUVEC) were purchased from the 655 (5 mL/mouse). Images of the FRET/CFP ratio as an index of Lonza Group, Ltd. These cell lines were acquired after 2012. PKA or ERK activity were prepared as described in the supple- Frozen stocks were prepared from initial stocks, and within mentary Materials and Methods (32). every 2 months, a new frozen stock was used for the experi- To visualize endothelial PKA activity under VEGF stimulation, ments. Colon-38 mouse colon cancer cells and 3LL mouse mouse VEGF164 (400 ng in 50 mL PBS) was injected intradermally lung carcinoma cells were provided by Drs. Setoyama and 30 minutes before observation. To examine PKA activity under the fi Chiba at Kyoto University (Kyoto, Japan) and Drs. Shime and speci c inhibitor treatment, H89 (5 mmol/L in 50 mLof5% Seya at Hokkaido University (Sapporo, Japan), respectively, DMSO/saline) or vehicle (5% DMSO/saline) was added to the and periodically authenticated by morphologic inspection. surface of Colon-38 tumors on the PKAchu mice. TumorornormalendothelialcellswereisolatedfromFRET mice as described previously (28). A pCX4 retroviral vector Vascular permeability assays (modified Miles assays) wasusedtoexpresstheKeimafluorescent protein or the Epac- Evans blue (10 mg/mL in PBS) was injected intravenously into cAMP sensor (29, 30). Cancer cells and HUVECs were main- tumor-bearing C57BL/6N Jcl mice at a concentration of 50 mg/kg tained in DMEM containing 10% fetal bovine serum and in with or without following reagents: dbcAMP (50 or 100 mg/kg), EGM-2MV media (Lonza), respectively. Following reagents 007 (10, 25, or 50 mg/kg), 6-Bnz (10, 25, or 50 mg/kg), or were used: Motesanib (Selleck Chemicals), Qtracker 655 Motesanib (50 mg/kg). Mice were subjected to PBS perfusion (Thermo Fisher ScientificInc.),N6,20-O-dibutyryladenosine 2 hours after the dye injection and sacrificed. The tumor and skin 30,50-cyclic monophosphate sodium salt (dbcAMP; Daiichi were then excised, dried at 60C for 24 hours, and weighed. The Sankyo Company, Ltd.), 8-(4-chlorophenylthio)-20–O-methy- dye was extracted from the tissues by incubation with 0.5 mL N,N- ladenosine 30,50-cyclic monophosphate (007) and N6-benzoy- dimethylformamide (Nacalai Tesque Inc.) at 56C for 48 hours. ladenosine-30,50-cyclic monophosphate (6-Bnz) (Biolog Life The dye was quantified by measuring the absorbance at 620 nm. Science Institute), Evans blue and isoflurane (Wako Pure The extracted dye was normalized to nanograms dye per milli- Chemicals, Ltd.), Mouse VEGF164 (BioLegend), H89 and gram tumor dry weight. In some experiments, 30 minutes after PD0325901 (Sigma-Aldrich Co. LLC). Evans blue injection, 400 ng mouse VEGF164 in 50 mL PBS was injected subcutaneously.

Mice and tumor implantation FRET imaging of PKA, ERK, or Epac activity in HUVEC We used PKAchu mice expressing the PKA FRET biosensor Lenti-X 293T cells (Takara Bio Inc.) were cotransfected with AKAR3EV-NES, PKA-NC mice expressing a negative control FRET the pCSIIbsr vector encoding the FRET biosensors AKAR3EV- biosensor AKAR3EV-NC, and Eisuke mice expressing the ERK NES or EKAREV-NES, psPAX2 (Addgene), and pCMV-VSV-G- FRET biosensor EKAREV-NES (26). FRET mice were back-crossed RSV-Rev by using 293fectin (Thermo Fisher ScientificInc.). more than nine generations to C57BL/6N Jcl mice (Japan SLC HUVECs were infected with the resulting lentivirus and cul- Inc.). Cancer cells (2 106 cells/50 mL PBS) were injected tured with EGM-2MV media on cover glass-bottomed dishes for subcutaneously into the flanks of mice. The animal protocols 16 to 24 hours before imaging. CFP and YFP images of HUVECs were reviewed and approved by the Animal Care and Use Com- were obtained by using an inverted microscope (LCV110; mittee of Kyoto University Graduate School of Medicine Olympus). Cells were stimulated with vehicle (PBS), VEGF164 (Nos. 12064, 13074, 14079, and 15064). (50–100 ng/mL), PD0325901 (10 mmol/L), and Forskolin (50 mmol/L)–IBMX (500 mmol/L) and analyzed as described Two-photon excitation microscopy and image processing previously (31). We used an FV1000MVE inverted microscope (Olympus Cor- poration) equipped with a 30 1.05 NA silicon-immersion Immunohistochemistry objective lens (UPLSAPO 30xS; Olympus), and an InSight Deep- Formalin-fixed tissue sections were incubated with primary See Ultrafast laser (0.95 W at 900 nm, Spectra Physics). The antibodies to CD31 (ad22538; Abcam), GFP (JL-8; Takara Bio excitation wavelength for cyan fluorescent protein (CFP) was Inc.), VEGFR2 (55B11; Cell Signaling Technology), or pVEGFR2 840 nm. For more information, refer to supplementary materials (19A10; Cell Signaling Technology) followed by incubation with and methods. Acquired images were processed and analyzed with secondary antibodies (Alexa Fluor; Thermo Fisher Scientific Inc.).

OF2 Cancer Res; 76(18) September 15, 2016 Cancer Research

Endothelial Hyperpermeability and Low PKA Activity in Tumors

Anti-CD31 antibody conjugated with Alexa Fluor 594 (BioLe- (Supplementary Fig. S2). H89 robustly reduced PKA activity gend), Hoechst33258, or DAPI (Sigma-Aldrich Co. LLC) was in stromal cells and slightly in the tumor endothelial cells also used. (Supplementary Fig. S2A and S2B). As expected, 6-Bnz, but not 007, activated PKA in tumor endothelial cells (Supplementary Statistical analysis Fig. S2C). All data were expressed as means standard deviation (SD). Statistical analysis was performed using GraphPad The PKA activity of intratumoral endothelial cells was lower Prism software (GraphPad Software Inc.). Student t test and than that of subcutaneous endothelial cells one-way ANOVA with Tukey post hoc analysis were used to Encouraged by the fi ndings obtained using Colon-38 cells, determine statistically significant differences. P values < 0.05 we extended our approach by using other cancer cells with were considered to indicate statistical significance. , P < 0.05; different morphological features (Fig. 2A). Because of the , P < 0.01; , P < 0.001. small cytoplasmic area of endothelial cells in the tangential image, the signal-to-noise ratios of FRET images were lower than those for the other cell types. Therefore, we time-lapse- Results imaged and quantified the PKA activity of at least 17 endo- In vivo imaging of tumor-bearing FRET mice visualized the PKA thelial cells of more than three mice under each condition (Fig. activity in the vascular endothelial cells of tumor and 2B). The PKA activity was significantly lower in the tumor subcutaneous tissue endothelial cells of all four cell lines examined—Colon-38 To understand the roles played by PKA in the hyperperme- (colon cancer), Panc-02 (pancreatic cancer), B16-F10 (mela- ability of intratumoral blood vessels, we compared the endo- noma), and 3LL (lung cancer)—than in all normal endothelial thelial PKA activities between normal subcutaneous blood cells of TBM and control mice. We did not find a significant vessels and intratumoral blood vessels. For visualization of the difference in the PKA activity among the endothelial cells of PKA activity in the tumor-bearing mice (TBM), we used PKAchu arterioles, capillaries, and venules. We also failed to detect any mice, which expressed a cytoplasmic FRET biosensor for PKA. difference between the subcutaneous endothelial cells in TBM The structure, mode of action, and stability of the FRET bio- and control mice. Notably, the densities of tumor cells, host sensors have been described previously (26, 33). Briefly, phos- cells, blood vessels, and collagen fibers were quite divergent phorylation of the substrate domain of the FRET biosensor among the tumors, strongly suggesting that the low PKA activity increases in the FRET signal, which can be quantified by the is a primary characteristic of the nascent intratumoral endo- intensity ratio of 530 nm FRET channel over 475 nm CFP thelial cells. To exclude the possibility that the difference in the channel (Supplementary Fig. S1A–S1C). Syngeneic Colon-38 FRET signal between the intratumoral and subcutaneous endo- colon cancer cells that expressed the Keima fluorescent protein thelial cells was caused by the difference in the physical prop- were subcutaneously implanted in the flanks of the PKAchu erties of tumor tissues and subcutaneous tissues, we performed mice. After tumor cells grew to palpable sizes, tumors and similar experiments with the PKA-NC mice, which expressed a subcutaneous tissues exposed by the skin-flap method were negative control PKA FRET biosensor lacking the PKA phos- observed under an inverted two-photon excitation microscope phorylation site in the same FRET biosensor backbone. No (Fig. 1A). In the tumor tissue, the FRET biosensor-expressing significant differences were observed between the intratumoral host cells, such as vascular endothelial cells and inflammatory and subcutaneous endothelial cells of PKA-NC mice (Fig. 2C). cells were clearly distinguished from the Keima-expressing These data strongly argued for the low intratumoral endothelial Colon-38 cancer cells (Fig. 1B, left). In the subcutaneous tissue PKA. of TBM, capillaries were clearly detected among the dense collagen fibers (Fig. 1B, right). No significant differences in ERK activities were observed The PKA activity of host cells is represented by FRET/CFP ratio between intratumoral and subcutaneous tissues images (Fig. 1C). The ratio range was fixed from 0.8 to 1.8 for We extended the same approach to Eisuke mice, which are consistency. As shown in the enlarged image, the PKA activity in transgenic mice expressing a FRET biosensor for ERK (Fig. 3 and the intratumoral endothelial cells was lower than the PKA activity Supplementary Fig. S1A and S1C). The distribution of ERK in the subcutaneous endothelial cells (Fig. 1C). The PKA activity of activity among different endothelial cells was not significantly neutrophils and platelets in the vessels could be conveniently differentfromthatofPKAactivityshowninFig.2.Neverthe- used as internal controls (Fig. 1C, arrows): The FRET/CFP values less, we failed to detect significant difference in ERK activity of neutrophils and platelets in the intratumoral blood vessel and between the intratumoral and subcutaneous endothelial cells. subcutaneous capillary were 1.37 0.15 (n ¼ 6) and 1.30 0.07 We also observed no difference between the TBM and control (n ¼ 6), respectively. We identified endothelial cells primarily by mice. These observations indicated that the difference between the location and shape of cells (Supplementary Fig. S1D). If tumor tissue and subcutaneous tissue was specificforPKA necessary, Z-stack images and images of Qtracker 655 vascular activity. tracer were used to distinguish endothelial cells from other cells such as pericytes (Supplementary Fig. S1E–S1G). Furthermore, we dbcAMP decreased the vascular permeability and increased the confirmed that the GFP-positive cells lining the internal surface of endothelial PKA activity in tumor tissues intratumoral blood vessels were positive for anti-CD31 antibody Previous studies have demonstrated that endothelial PKA is a (Fig. 1D). key player for endothelial barrier function (18, 19). It is also To verify the specificityofthebiosensor,weperformed known that vascular permeability is high in intratumoral blood in vivo FRET imaging with a PKA inhibitor, H89, a PKA-specific vessels. Therefore, we hypothesized that the low PKA activity in agonist, 6-Bnz, or an Epac/cAMP-GEF specific-agonist, 007 the intratumoral endothelial cells is responsible for the high

www.aacrjournals.org Cancer Res; 76(18) September 15, 2016 OF3

Yamauchi et al.

A Cancer cells Drugs Tumor Skin flap 20–30 days

FRET Mouse Tumor-bearing mouse Objective lens (1) Implantation of cancer cells (2) In vivo FRET imaging (3) Administration of drugs

B Tumor tissue Subcutaneous tissue (TBM) D Tumor tissue

* V * V V V Colon-38 cells

Nucleus * V GFP Host cells, Collagen

* C Intratumoral blood vessels Subcutaneous capillary (TBM)

1.8 L Nucleus L CD31 0.8 PKA ActivityPKA (FRET/CFP)

Figure 1. Invivo imaging of FRET mice visualized PKA activities in the endothelial cells within tumor tissue and subcutaneous tissue. A, experimental schemes: 1, Colon-38 colon cancer cells expressing the Keima fluorescent protein were injected subcutaneously into PKAchu mice, which expressed a cytoplasmic FRET biosensor for PKA. 2, after the cancer cells had grown to visible size, intratumoral or subcutaneous (s.c.) blood vessels were observed by the skin-flap method under an inverted two-photon excitation microscope. 3, if necessary, drugs were administrated intravenously (i.v.) during imaging. B, tumor tissue and subcutaneous tissue were imaged for collagen fiber (second harmonic, blue), host cells (YFP, green), and Colon-38 tumor cells (Keima, red). V, blood vessels. Scale bar, 50 mm. C, corresponding images of FRET/CFP representing PKA activity in the host cells of the regions shown by white boxes in B. The FRET/CFP images are shown in intensity- modulated display mode with a 32-intensity in 8-ratio (0.8–1.8). Arrowheads, arrows, and L indicate endothelial cells, platelets, and the luminal space of blood vessels, respectively. Scale bar, 20 mm. D, the tumor tissue was immunostained for GFP (green) and CD31 (red). Arrowheads show GFP-positive endothelial cells that formed the inner cellular lining of intratumoral blood vessels (V). Asterisks indicate GFP-negative cancer cells. Nuclei were stained with Hoechst (blue). Scale bar, 50 mm.

vascular permeability in the tumor. To validate this hypothesis, we skin, confirming the hyperpermeability of the tumor vessels conducted a modified Miles assay for measuring vascular perme- (Fig. 4B). In the dbcAMP-injected mice, however, the extrava- ability in the presence or absence of a cAMP analogue, dbcAMP sation of Evans blue was as low as that in the skin. Importantly, (Fig. 4A). the effect of dbcAMP was not detectable in the skin, strongly Colon-38 or Panc-02 cells were injected into the flanks of suggesting that the PKA activity in the normal subcutaneous C57BL/6N Jcl mice to prepare TBM. After intravenous injection endothelium is sufficiently high to prevent extravasation of of Evans blue to TBM, the dye extravasation from the tumor and Evans blue. Similar results were obtained with the Panc-02 the skin was quantified at 2 hour after administration (Fig. 4A). As tumor model. The effect of administered dbcAMP on the expected, in the PBS-injected Colon-38-bearing mice, the extrav- endothelial PKA was validated by in vivo FRET imaging (Fig. asation of Evans blue dye was higher in the tumor than the 4C). dbcAMP robustly activated PKA in both tumor and normal

OF4 Cancer Res; 76(18) September 15, 2016 Cancer Research

Endothelial Hyperpermeability and Low PKA Activity in Tumors

A Intratumoral blood vessels Subcutaneous Colon-38 Panc-02 B16-F10 3LL arteriole (TBM)

V V V V V Host cells Cancer cells Collagen

1.8 PKA Activity Activity PKA (FRET/CFP) 0.8

Subcutaneous Subcutaneous Subcutaneous Mesenteric Hepatic venule (TBM) capillary (TBM) capillary capillary sinusoid

V V V V V Host cells Collagen

1.8

PKA Activity Activity PKA (FRET/CFP) 0.8

B C 2.2 2.2 *** *** * ***

1.8 1.8

1.4 1.4 n.s. in PKA-NC mice in PKA-NC

in endothelial cells 1.0 1.0 PKA Activity (FRET/CFP) Activity PKA FRET/CFP of endothelial cells FRET/CFP of

0.6 0.6

Intratumoral blood Subcutaneous Intratumoral blood vessels (TBM) vessels

Figure 2. PKA activity was lower in the intratumoral endothelial cells than the subcutaneous endothelial cells. A, TBM were prepared as in Fig. 1. Top, tumor or normal tissues were imaged for collagen fiber (second harmonic, blue), host cells (YFP, green), and tumor cells (Keima, red). V, blood vessels. Bottom, corresponding images of FRET/CFP representing PKA activity in the host cells. The tumor cells used here were Colon-38 colon cancer cells, Panc-02 pancreatic cancer cells, B16-F10 melanoma cells, and 3LL lung cancer cells (n > 3 mice for each). In subcutaneous arteriole, perictyes could also be observed (arrows). Scale bar, 20 mm. B, endothelial cells were randomly selected and examined for their PKA activity. Results obtained from at least three mice are combined. Black dots and red bars indicate PKA activity in each endothelial cell (n ¼ 17–40 in each cell) and the mean SD, respectively. Differences from normal endothelial cells were evaluated by one-way ANOVA with Tukey post hoc analysis: , P < 0.05; , P < 0.001. Images of all endothelial cells except those of intratumoral endothelial cells were pooled and used as the reference. C, FRET/CFP of normal and tumor endothelial cells in transgenic mice expressing the negative control FRET biosensor (PKA-NC). Results obtained from three mice are combined. Black dots and red bars indicate the FRET/CFP in each endothelial cell (n ¼ 22–24 in each cell) and the mean SD, respectively. No significant difference in FRET/CFP was observed. n.s., not significant (Student t test). www.aacrjournals.org Cancer Res; 76(18) September 15, 2016 OF5

Yamauchi et al.

A B Intratumoral Subcutaneous n.s. blood vessels capillary (TBM) n.s. 2.5

V 2.0 V Collagen Host cells Colon-38 cells 1.5

in endothelial cells 1.0

2.0 ERK Activity (FRET/CFP)

0.5

0.8

ERK Activity ERK Activity (FRET/CFP) Intratumoral Subcutaneous

Figure 3. Intratumoral and subcutaneous endothelial cells exhibited similar ERK activity. TBMs were prepared as in Fig. 1 using Eisuke mice, which express a FRET biosensor for ERK. A, top, Colon-38 tumor tissue and subcutaneous tissue were imaged for collagen ﬁber (second harmonic, blue), host cells (YFP, green), and tumor cells (Keima, red). V, blood vessels. Bottom, corresponding images of FRET/CFP representing ERK activity in the host cells (n ¼ 3 mice for each). Scale bar, 50 mm. B, basal ERK activities of normal and tumor endothelial cells. Endothelial cells were randomly selected in the CFP images and examined for their ERK activity in the corresponding FRET/CFP ratio image. Results obtained from three mice are combined. Black dots and red bars indicate the ERK activity in each endothelial cell (n ¼ 21–24 in each cell) and the mean SD, respectively. n.s., not signiﬁcant (one-way ANOVA).

endothelial cells (Fig. 4D). Taken together, these data indicate shown to play key roles in vascular permeability and angio- that tumor vascular permeability is in part dependent on the genesis in tumors (6). To examine the role of VEGFR signaling low endothelial PKA activity. on PKA, we tested the effect of a VEGFR inhibitor on tumor vascular permeability and endothelial PKA activity by Miles Not only PKA but also Epac/cAMP-GEF reduced tumor vascular assay and in vivo FRET imaging, respectively. Among the permeability available VEGFR kinase inhibitors, we chose Motesanib based cAMP can regulate vascular permeability by PKA and Epac/ on its high water solubility. Mice were injected intravenously cAMP-GEF. To assess the contribution of PKA and Epac/cAMP- with Evans blue dye and Motesanib (50 mg/kg) and subjected GEF to the cAMP-induced decrease of tumor vascular perme- to the Miles assay. Motesanib reduced the extravasation of ability, we used a PKA-specific agonist, 6-Bnz, and an Epac/ Evans blue in the tumor, but not the skin, indicating that cAMP-GEF-specificagonist,007.Micewereinjectedintrave- Motesanib inhibited tumor vascular permeability (Fig. 6A). nously together with Evans blue dye and the agonists (10, 25, This findingisconsistentwithpreviousstudiesthatdemon- and50mg/kg)andsubjectedtoaMilesassay(Fig.5).Both strated an inhibition of VEGF-induced vascular permeability 6-Bnz and 007 decreased the extravasation of Evans blue in a by Motesanib administration (35). By immunohistochemistry, dose-dependent manner. Neither 6-Bnz nor 007 significantly we confirmed that Motesanib suppressed the VEGFR2 tyrosine affected the extravasation of Evans blue in the skin. Although kinase activity, but not the expression of VEGFR2 (Supple- we did not test the cAMP levels in the endothelial cells, these mentary Fig. S3). data strongly suggested that the low cAMP level in tumor We next examined the response of endothelial PKA activity to endothelial cells at least partially contributed to the high Motesanib by in vivo FRET imaging. Motesanib robustly activated vascular permeability in tumor tissues. PKA in the endothelial cells of the tumors, but not in the endothelial cells of the subcutaneous tissues (Fig. 6B), indicating Inhibition of VEGFR activated tumor endothelial PKA and that VEGFR played a major role in the suppression of PKA. reduced vascular permeability in tumor tissue Notably, Motesanib had no effect on the ERK activities in tumor The cAMP level and PKA activity are regulated by numerous and normal endothelial cells (Fig. 6C). The lack of effect on ERK signaling molecules, including Gs-coupled GPCRs and phos- activity is probably due to the low basal ERK activity. In fact, when phodiesterases (34). Therefore, rather than examining the ERK activity in tumor endothelial cells was elevated by VEGF, contribution of direct regulators of cAMP, we focused on Motesanib markedly decreased ERK activity (Supplementary VEGFs and their cognate receptors (VEGFRs), which have been Fig. S4A–S4C).

OF6 Cancer Res; 76(18) September 15, 2016 Cancer Research

Endothelial Hyperpermeability and Low PKA Activity in Tumors

A Cancer cells Evans blue + dbcAMP Skin Tumor

14-21 days 2 h

C57BL/6N Jcl mouse Tumor-bearing mouse (1) Implantation of tumor cells (2) Dye and dbcAMP injection (3) Extravasation of Evans blue

B PBS C PKA Activity (FRET/CFP) dbcAMP (50 mg/kg) 0.6 1.8 (min) D dbcAMP (100 mg/kg) *** ** –4 LL *** 1.4 ** *** ** 0 200 200 1.3 ** 4 1.2

150 150 8 1.1 12

(FRET/CFP) 1.0 100 100 16

Normalized PKA activity Normalized PKA 0.9 20

(ng/mg tissue in 2 h) 0.8 50 50 Extravasation of Evans blue Evans Extravasation of 24

28 0 0 Intratumoral Subcutaneous Colon-38 Skin Panc-02 Skin PBS dbcAMP

Figure 4. dbcAMP reduced tumor vascular permeability. A, experimental scheme for a modiﬁed Miles assay: 1, cancer cells were injected subcutaneously into C57BL/6 Jcl mice. 2, vehicles or drugs were intravenously administrated with Evans blue (50 mg/kg). 3, two hours later, mice were sacriﬁced for the examination of Evans blue extravasation. B, PBS or dbcAMP (50 or 100 mg/kg) was injected to examine the Evans blue extravasation. At least three mice were examined in each group. Data are represented as the mean SD (n ¼ 5–14 in each tissue). Differences were evaluated by the Student t test: , P < 0.01; , P < 0.001. C, time-lapse FRET images of the tumor endothelial cells in the PKAchu mice bearing Colon-38 tumors. A vehicle (PBS) or dbcAMP (50 mg/kg) was injected intravenously at 0 minute. Arrowheads and L indicate endothelial cells and the luminal space of blood vessels, respectively. Scale bar, 10 mm. D, PKA activities in the intratumoral and subcutaneous endothelial cells. FRET images were acquired 16 minutes after vehicle (PBS) or dbcAMP injection (50 mg/kg). Three mice were analyzed for each condition (n ¼ 7–10 in each cell). Endothelial cells were randomly selected and examined for their PKA activity. Data are represented as the PKA activity (FRET/CFP) normalized to the respective mean values before drug injection. Bars, mean SD. Differences from the vehicle group were evaluated by Student t test. , P < 0.01; , P < 0.001.

VEGF decreased endothelial PKA activity and increased vascular cells (HUVEC) expressing the PKA FRET biosensor. In stark permeability in vivo contrast to our in vivo observations, we found that PKA activity To confirm the role of PKA on the VEGF-induced increase in was increased rapidly in HUVECs (Fig. 7D and E). The increase in vascular permeability, PKA activity and vascular permeability the cAMP level was also confirmed by the Epac-cAMP sensor were measured in the VEGF-administered skin. Evans blue was (Supplementary Fig. S5; ref. 29). To examine if the discrepancy intravenously injected and 30 minutes later, PBS or VEGF164 was caused by the in vitro experimental condition, we isolated was injected intradermally. VEGF increased the vascular per- intratumoral and subcutaneous endothelial cells from the trans- meability in the skin (Fig. 7A). In a parallel experiment, PBS genic and repeated the experiments (Supplementary Fig. S6). In or VEGF164 was intradermally injected into PKAchu mice, contrast to HUVECs and similarly to the subcutaneous endo- followed by in vivo FRET imaging for PKA activity (Fig. 7B). As thelial cells, PKA activity in the isolated subcutaneous endo- shown in Fig. 7C, the PKA activity in the capillary endothelial thelial cells was higher than the isolated intratumoral endo- cells was significantly lower in the VEGF-injected skin than in thelial cells and decreased by VEGF stimulation. These results the PBS-injected skin. Taken together, these data strongly suggest that the isolated primary cells are more similar to the suggested that the VEGFR signaling pathway increases vascular vascular endothelial cells in vivo than to the cultured HUVECs. permeability in part by reducing the PKA activity in endothe- This is likely due to the difference in proliferation ability lial cells. between the two types of normal endothelial cells. In fact, the Finally, we attempted to recapitulate the VEGF-induced isolated endothelial cells ceased proliferation and became decrease of PKA activity in human umbilical vascular endothelial senescent several days after isolation.

www.aacrjournals.org Cancer Res; 76(18) September 15, 2016 OF7

Yamauchi et al.

Colon-38 Skin 200 200 Figure 5. Activation of PKA or Epac/cAMP-GEF 150 150 reduced tumor vascular permeability. Mice bearing Colon-38 tumors were treated * ** * ** with the PKA activator 6-Bnz or Epac/ 100 100 cAMP-GEF activator 007 (0, 10, 25, or 50 mg/kg) together with Evans blue. Tumors and skin were excised 2 hours 50 50 after administration for the analysis of

(ng/mg tissue in 2 h) Evans blue extravasation. At least three mice were examined in each group. Data, Extravasation of Evans blue Evans Extravasation of 0 0 mean SD (n ¼ 6–14 in each tissue). 6-Bnz 0 502510 000 0 10 5025 000 Differences from the nontreated group were evaluated by the Student t test: 007 0000 502510 0000 502510 , P < 0.05; , P < 0.01. Injection dose (mg/kg) Injection dose (mg/kg)

Discussion activity may be responsible for the intratumoral vascular hyper- Intratumoral vascular hyperpermeability regulates tumor pro- permeability. Due to the technical difficulties, we needed to gression, tumor metastasis, and intratumoral drug delivery (5). observe implanted cancer cells that grow faster than naturally- Although inhibition of PKA is known to increase vascular per- occurring cancer cells. It awaits further study whether this property meability in normal tissue (36), the role of PKA in the intratu- is a common hallmark of cancer cells. moral vascular hyperpermeability has not been clarified. Our VEGF-mediated activation of endothelial VEGFRs is a canonical in vivo FRET imaging has implied that the basal PKA activity is pathway to increase vascular permeability in diseased tissues, significantly lower in the intratumoral endothelial cells than in including tumors (35, 37). Accordingly, we observed that VEGF the subcutaneous endothelial cells (Fig. 2B) and that the low PKA increased the vascular permeability of subcutaneous tissue

A B *** n.s. C n.s. n.s. 200 *** Vehicle 1.4 1.4 Motesanib 1.3 1.3 150 1.2 1.2

100 1.1 1.1 (FRET/CFP) 1.0 (FRET/CFP) 1.0

(ng/mg tissue in 2 h) 50 Normalized ERK activity activity Normalized ERK Normalized PKA activity Normalized PKA 0.9 0.9 Extravasation of Evans blue Evans Extravasation of 0.8 0.8 0 Colon-38 Skin 0.7 0.7

Intratumoral Subcutaneous Intratumoral Subcutaneous

Figure 6. The VEGFR inhibitor Motesanib reduced the tumor vascular permeability, and this reduction was mediated by endothelial PKA activation. A, TBMs were treated with vehicle (saline containing 5% DMSO) or Motesanib (50 mg/kg) together with Evans blue. The tumors and skins were excised 2 hours after administration, and Evans blue extravasation was quantiﬁed and expressed as ng of dye/mg of tissue (ng/mg tissue; n 3). Data, mean SD (n ¼ 5–8 in each tissue). Differences from the vehicle group were evaluated by the Student t test: , P < 0.001. Scale bar, 10 mm. B and C, PKA or ERK activities in the intratumoral and subcutaneous endothelial cells were imaged in the PKAchu mice or Eisuke mice bearing Colon-38 tumors as in Fig. 1. FRET images were acquired 26 minutes after the vehicle or Motesanib injection (50 mg/kg) (N ¼ 3). Endothelial cells were randomly selected and examined for their PKA or ERK activity. Data are represented as the activity (FRET/CFP) normalized to the respective mean values before drug injection. Bars, mean SD (n ¼ 6–12 in each cell). Differences from the vehicle group were evaluated by the Student t test: , P < 0.001. n.s., not signiﬁcant by the Student t test.

OF8 Cancer Res; 76(18) September 15, 2016 Cancer Research

Endothelial Hyperpermeability and Low PKA Activity in Tumors

A * B C ** Subcutaneous capillary 200 2.2

2.0 150

PBS 1.8 Collagen Host cells 100 VEGF 1.6

1.8 (ng/mg tissue) 50 1.4 PKA Activity (FRET/CFP) Activity PKA

Extravasation of Evans blue Evans Extravasation of 1.2

0 in subcutaneous endothelial cells 0.8 Skin 1.0 PBS VEGF

PKA Activity (FRET/CFP) Activity PKA PBS VEGF

PBS VEGF E PKA Activity 1.8 (FRET/CFP) D HUVEC PBS

PKA Activity(FRET/CFP) VEGF 0.6 PBS 1.8 (min) –8 0 8 16 24 32 40

*** F 1.3

0.6 1.2 VEGF 1.1

1.0

0.9 Before After HUVEC (FRET/CFP) in (–8 min) (8 min) activity Normalized PKA PBS VEGF

Figure 7. VEGF enhanced vascular permeability and decreased PKA activity in the subcutaneous endothelial cells. A, extravasation of Evans blue in skin was quantified 30 minutes after subcutaneous injection of PBS (vehicle) or VEGF into C57BL/6N Jcl mice. The mean SD values are shown (n ¼ 3 in each tissue). Differences from the vehicle group were evaluated by the Student t test: , P < 0.05. The photographs on the bottom indicate dye extravasation of Evans blue in skin. B, PKA activity in the subcutaneous endothelial cells was visualized with PKAchu mice as in Fig. 1. Mice were treated with PBS or VEGF for 30 minutes before acquisition of the images. Scale bar, 10 mm. C, endothelial cells were randomly selected and examined for their PKA activity (N ¼ 3). Black dots and red bars indicate the PKA activity in each endothelial cell (n ¼ 27–29 in each cell) and the mean SD, respectively. Differences from the vehicle group (PBS) were evaluated by the Student t test: , P < 0.01. D–F, time lapse FRET images of HUVEC expressing the PKA biosensor. At 0 minute, 50 ng/mL of VEGF164 or PBS was added to the cells and imaged every 4 minutes for 1 hour. The whole viewfield images (D) and images of a representative cell (E) are shown. Scale bar, 10 mm. The experiment was repeated three times with similar results. The normalized PKA activity (FRET/CFP) was quantified at 32 minutes for 18 cells randomly selected from each of the three different experiments (F). Bars are the mean SD. Differences from the vehicle group were evaluated by the Student t test. , P < 0.001.

(Fig. 7A), while a VEGFR inhibitor, Motesanib, reduced the (39); therefore, when VEGF is secreted by the interstitial cells, vascular permeability of tumor tissue (Fig. 6A). Importantly, VEGFR2, but not VEGFR1, will primarily transduce signals in the under both conditions, PKA activity in the endothelial cells was diseased tissues. VEGFR2 may suppress, rather than activate, PKA inversely correlated with the vascular permeability. In contrast to in vivo. In contrast, VEGF will stimulate both VEGFR1 and VEGFR2 our observations, Xiong and colleagues have shown that VEGF in vitro and activate PKA. increases cAMP and activates PKA in HUVECs (38). We have also How is PKA activity regulated in the normal and intratu- conﬁrmed that VEGF activates PKA in HUVECs (Fig. 7D and E). In moral endothelial cells? In most cell types, PKA activity vivo environmental factors, such as cytokines, growth factors, depends exclusively on the cAMP level (40). Therefore, the extracellular matrix, or 3D structures including an interaction endothelial PKA activity may be regulated primarily by the with pericytes, may account for this discrepancy between the activity of G protein-coupled receptors (GPCR) associated with in vivo and in vitro observations. For example, two VEGFR-family either Gs or Gi. Previous studies have demonstrated speciﬁc proteins, VEGFR1 and VEGFR2, are localized at the apical and expression of Gi coupled receptors such as the Apelin receptor basolateral surfaces of endothelial cells, respectively, in the tissues (41) and sphingosine-1-phosphate receptors (42) in tumor

www.aacrjournals.org Cancer Res; 76(18) September 15, 2016 OF9

Yamauchi et al.

endothelial cells. An endogenous PKA inhibitor (PKI) may also cells. Nevertheless, we conclude that in vivo FRET imaging is a be involved in the regulation of PKA (16). It is important to powerful approach to fill the gap between in vitro signal trans- note that not only the authentic ligand to GPCR but also other duction research and in vivo cancer biology, if we understand the receptors, such as VEGFR, can regulate the activity of GPCR. limitation of this technology. Needless to say, we cannot exclude the possible involvement of other cAMP regulators from the regulation of the cAMP level Disclosure of Potential Conflicts of Interest and PKA activity in the endothelial cells. For example, phos- No potential conflicts of interest were disclosed. phodiesterases have been shown to regulate the cAMP level and PKA activity under various conditions (34). Further studies will be required to elucidate the signaling pathway by which cAMP Authors' Contributions and PKA activity in the intratumoral endothelial cells are Conception and design: F. Yamauchi, Y. Kamioka, T. Yano, M. Matsuda controlled and in turn control the permeability of endothelial Development of methodology: F. Yamauchi, Y. Kamioka, M. Matsuda Acquisition of data (provided animals, acquired and managed patients, cells. provided facilities, etc.): F. Yamauchi, Y. Kamioka cAMP stabilizes the endothelial barrier and antagonizes the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, cytokine-mediated increase of vascular permeability (17, 20). To computational analysis): F. Yamauchi, M. Matsuda examine which cAMP effector plays a principal role in the main- Writing, review, and/or revision of the manuscript: F. Yamauchi, Y. Kamioka, tenance of endothelial barrier function, we used specific activators M. Matsuda for PKA and Epac/cAMP-GEF. Unexpectedly, we found that both Administrative, technical, or material support (i.e., reporting or organizing fi fi data, constructing databases): F. Yamauchi, Y. Kamioka, M. Matsuda the PKA-speci c activator 6-Bnz and the Epac/cAMP-GEF-speci c Study supervision: Y. Kamioka, T. Yano, M. Matsuda activator 007 decreased the vascular permeability of tumor tissues (Fig. 5), suggesting that the endothelial barrier is maintained by both PKA and Epac/cAMP-GEF in vivo. Our results also under- Acknowledgments scored that in vivo FRET imaging can help not only to clarify We are grateful to Kees Jalink for Epac cAMP-sensor, Shigetomo Fukuhara for insightful suggestions and the members of the Matsuda Laboratory for their biological processes, but also in the search for new strategies of helpful input, Y. Inaoka, K. Hirano, K. Takakura, and A. Kawagishi for their anticancer therapy. Previous studies demonstrated that inhibition technical assistance, and Medical Research Support Center of Kyoto University of tumor vascular permeability could lead to suppression of for in vivo imaging. tumor growth (43) and improvement of chemotherapy (44). Taken these observations into account, by restoring PKA activity Grant Support in the intratumoral endothelial cells to the level in normal M. Matsuda was funded by the Innovative Techno-hub for Integrated endothelial cells, the intratumoral vascular hyperpermeability Medical Bio-imaging Project of MEXT, by the Platform Project for Supporting may be alleviated and thereby control tumor growth. in Drug Discovery and Life Science Research (Platform for Dynamic Approaches A drawback of the in vivo FRET imaging is that validation of the to Living System) of MEXT and AMED, Japan, by a Grant-in-Aid for Scientific specificity is not as easy as in studies by using tissue culture cells. Research on Innovative Areas "Resonance Biology" of MEXT, and by the Naito For example, we cannot exclude the possibility that the PKA Foundation. The costs of publication of this article were defrayed in part by the payment of biosensor is also phosphorylated by other kinases, unless we use advertisement fi page charges. This article must therefore be hereby marked in PKA knockout mice. Similarly, because of the speci city issue accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (45), the H89-induced decrease in FRET signal (Supplementary Fig. S2) may not be sufficient for excluding the involvement of Received December 26, 2015; revised June 13, 2016; accepted July 4, 2016; other kinases in the high basal FRET signal in normal endothelial published OnlineFirst August 3, 2016.

References 1. McDonald DM, Baluk P. Signiﬁcance of blood vessel leakiness in cancer. 10. Roberts WG, Palade GE. Increased microvascular permeability and endo- Cancer Res 2002;62:5381–5. thelial fenestration induced by vascular endothelial growth factor. J Cell Sci 2. Gerlowski LE, Jain RK. Microvascular permeability of normal and neo- 1995;108(Pt 6):2369–79. plastic tissues. Microvasc Res 1986;31:288–305. 11. Feng D, Nagy JA, Brekken RA, Pettersson A, Manseau EJ, Pyne K, et al. 3. Maeda H, Fang J, Inutsuka T, Kitamoto Y. Vascular permeability enhance- Ultrastructural localization of the vascular permeability factor/vascular ment in solid tumor: various factors, mechanisms involved and its impli- endothelial growth factor (VPF/VEGF) receptor-2 (FLK-1, KDR) in normal cations. Int Immunopharmacol 2003;3:319–28. mouse kidney and in the hyperpermeable vessels induced by VPF/VEGF- 4. Mehta D, Malik AB. Signaling mechanisms regulating endothelial perme- expressing tumors and adenoviral vectors. J Histochem Cytochem ability. Physiol Rev 2006;86:279–367. 2000;48:545–56. 5. Azzi S, Hebda JK, Gavard J. Vascular permeability and drug delivery in 12. Hobbs SK, Monsky WL, Yuan F, Roberts WG, Grifﬁth L, Torchilin cancers. Front Oncol 2013;3:211. VP, et al. Regulation of transport pathways in tumor vessels: role of 6. Weis SM, Cheresh DA. Pathophysiological consequences of VEGF-induced tumor type and microenvironment. Proc Natl Acad Sci U S A vascular permeability. Nature 2005;437:497–504. 1998;95:4607–12. 7. Jain RK. Antiangiogenesis strategies revisited: from starving tumors to 13. Hippenstiel S, Krull M, Ikemann A, Risau W, Clauss M, Suttorp N. VEGF alleviating hypoxia. Cancer Cell 2014;26:605–22. induces hyperpermeability by a direct action on endothelial cells. Am J 8. Yuan F, Dellian M, Fukumura D, Leunig M, Berk DA, Torchilin Physiol 1998;274:L678–84. VP, et al. Vascular permeability in a human tumor xenograft: 14. Gavard J, Gutkind JS. VEGF controls endothelial-cell permeability by molecular size dependence and cutoff size. Cancer Res 1995;55: promoting the beta-arrestin-dependent endocytosis of VE-cadherin. Nat 3752–6. Cell Biol 2006;8:1223–34. 9. Jain RK. Normalizing tumor vasculature with anti-angiogenic therapy: a 15. Monsky WL, Fukumura D, Gohongi T, Ancukiewcz M, Weich HA, Torchilin new paradigm for combination therapy. Nat Med 2001;7:987–9. VP, et al. Augmentation of transvascular transport of macromolecules and

OF10 Cancer Res; 76(18) September 15, 2016 Cancer Research

Endothelial Hyperpermeability and Low PKA Activity in Tumors

nanoparticles in tumors using vascular endothelial growth factor. Cancer 30. Akagi T, Shishido T, Murata K, Hanafusa H. v-Crk activates the phosphoi- Res 1999;59:4129–35. nositide 3-kinase/AKT pathway in transformation. Proc Natl Acad Sci U S A 16. Lum H, Jaffe HA, Schulz IT, Masood A, RayChaudhury A, Green RD. 2000;97:7290–5. Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protec- 31. Aoki K, Matsuda M. Visualization of small GTPase activity with fluores- tion to endothelial barrier dysfunction. Am J Physiol 1999;277:C580–8. cence resonance energy transfer-based biosensors. Nat Protoc 2009; 17. Irie K, Fujii E, Ishida H, Wada K, Suganuma T, Nishikori T, et al. Inhibitory 4:1623–31. effects of cyclic AMP elevating agents on lipopolysaccharide (LPS)-induced 32. Mizuno R, Kamioka Y, Kabashima K, Imajo M, Sumiyama K, Nakasho E, microvascular permeability change in mouse skin. Br J Pharmacol et al. In vivo imaging reveals PKA regulation of ERK activity during 2001;133:237–42. neutrophil recruitment to inflamed intestines. J Exp Med 2014;211: 18. Waschke J, Drenckhahn D, Adamson RH, Barth H, Curry FE. cAMP protects 1123–36. endothelial barrier functions by preventing Rac-1 inhibition. Am J Physiol 33. Komatsu N, Aoki K, Yamada M, Yukinaga H, Fujita Y, Kamioka Y, et al. Heart Circ Physiol 2004;287:H2427–33. Development of an optimized backbone of FRET biosensors for kinases 19. Kobayashi K, Tsubosaka Y, Hori M, Narumiya S, Ozaki H, Murata T. and GTPases. Mol Biol Cell 2011;22:4647–56. Prostaglandin D2-DP signaling promotes endothelial barrier function via 34. Surapisitchat J, Beavo JA. Regulation of endothelial barrier function by the cAMP/PKA/Tiam1/Rac1 pathway. Arterioscler Thromb Vasc Biol cyclic nucleotides: the role of phosphodiesterases. Handb Exp Pharmacol 2013;33:565–71. 2011:193–210. doi: 10.1007/978-3-642-17969-3_8 20. Fukuhara S, Sakurai A, Sano H, Yamagishi A, Somekawa S, Takakura N, 35. Polverino A, Coxon A, Starnes C, Diaz Z, DeMelfi T, Wang L, et al. AMG 706, et al. Cyclic AMP potentiates vascular endothelial cadherin-mediated cell- an oral, multikinase inhibitor that selectively targets vascular endothelial cell contact to enhance endothelial barrier function through an Epac-Rap1 growth factor, platelet-derived growth factor, and kit receptors, potently signaling pathway. Mol Cell Biol 2005;25:136–46. inhibits angiogenesis and induces regression in tumor xenografts. Cancer 21. Adamson RH, Ly JC, Sarai RK, Lenz JF, Altangerel A, Drenckhahn D, Res 2006;66:8715–21. et al. Epac/Rap1 pathway regulates microvascular hyperpermeability 36. Birukova AA, Burdette D, Moldobaeva N, Xing J, Fu P, Birukov KG. Rac induced by PAF in rat mesentery. Am J Physiol Heart Circ Physiol GTPase is a hub for protein kinase A and Epac signaling in endothelial 2008;294:H1188–96. barrier protection by cAMP. Microvasc Res 2010;79:128–38. 22. Brown EB, Campbell RB, Tsuzuki Y, Xu L, Carmeliet P, Fukumura D, et al. In 37. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. vivo measurement of gene expression, angiogenesis and physiological Nat Med 2003;9:669–76. function in tumors using multiphoton laser scanning microscopy. Nat 38. Xiong Y, Huo Y, Chen C, Zeng H, Lu X, Wei C, et al. Vascular endothelial Med 2001;7:864–8. growth factor (VEGF) receptor-2 tyrosine 1175 signaling controls VEGF- 23. Harney AS, Arwert EN, Entenberg D, Wang Y, Guo P, Qian BZ, et al. Real- induced von Willebrand factor release from endothelial cells via phos- time imaging reveals local, transient vascular permeability, and tumor cell pholipase C-gamma 1- and protein kinase A-dependent pathways. J Biol intravasation stimulated by TIE2hi macrophage-derived VEGFA. Cancer Chem 2009;284:23217–24. Discov 2015;5:932–43. 39. Hudson N, Powner MB, Sarker MH, Burgoyne T, Campbell M, Ockrim ZK, 24. Hirata E, Girotti MR, Viros A, Hooper S, Spencer-Dene B, Matsuda M, et al. et al. Differential apicobasal VEGF signaling at vascular blood-neural Intravital imaging reveals gow BRAF inhibition generates drug-tolerant barriers. Dev Cell 2014;30:541–52. microenvironments with high integrin beta1/FAK signaling. Cancer Cell 40. Kim C, Vigil D, Anand G, Taylor SS. Structure and dynamics of PKA 2015;27:574–88. signaling proteins. Eur J Cell Biol 2006;85:651–4. 25. Nobis M, McGhee EJ, Morton JP, Schwarz JP, Karim SA, Quinn J, et al. 41. Kidoya H, Kunii N, Naito H, Muramatsu F, Okamoto Y, Nakayama T, et al. Cancer ResIntravital FLIM-FRET imaging reveals dasatinib-induced spatial The apelin/APJ system induces maturation of the tumor vasculature and control of Src in pancreatic cancer. Cancer Res 2013;73:4674–86. improves the efficiency of immune therapy. Oncogene 2012;31:3254–64. 26. Kamioka Y, Sumiyama K, Mizuno R, Sakai Y, Hirata E, Kiyokawa E, et al. 42. Chae SS, Paik JH, Furneaux H, Hla T. Requirement for sphingosine 1- Live imaging of protein kinase activities in transgenic mice expressing FRET phosphate receptor-1 in tumor angiogenesis demonstrated by in vivo RNA biosensors. Cell Strcut Funct 2012;37:65–73. interference. J Clin Invest 2004;114:1082–9. 27. Johnsson AK, Dai Y, Nobis M, Baker MJ, McGhee EJ, Walker S, et al. The Rac- 43. Gratton JP, Lin MI, Yu J, Weiss ED, Jiang ZL, Fairchild TA, et al. Selective FRET mouse reveals tight spatiotemporal control of Rac activity in primary inhibition of tumor microvascular permeability by cavtratin blocks tumor cells and tissues. Cell Rep 2014;6:1153–64. progression in mice. Cancer Cell 2003;4:31–9. 28. Hida K, Hida Y, Amin DN, Flint AF, Panigrahy D, Morton CC, et al. Tumor- 44. Turley RS, Fontanella AN, Padussis JC, Toshimitsu H, Tokuhisa Y, Cho EH, associated endothelial cells with cytogenetic abnormalities. Cancer Res et al. Bevacizumab-induced alterations in vascular permeability and drug 2004;64:8249–55. delivery: a novel approach to augment regional chemotherapy for in-transit 29. Ponsioen B, Zhao J, Riedl J, Zwartkruis F, van der KG, Zaccolo M, et al. melanoma. Clin Cancer Res 2012;18:3328–39. Detecting cAMP-induced Epac activation by fluorescence resonance 45. Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, et al. The energy transfer: Epac as a novel cAMP indicator. EMBO Rep 2004; selectivity of protein kinase inhibitors: a further update. Biochem J 5:1176–80. 2007;408:297–315.

www.aacrjournals.org Cancer Res; 76(18) September 15, 2016 OF11

In Vivo FRET Imaging of Tumor Endothelial Cells Highlights a Role of Low PKA Activity in Vascular Hyperpermeability

Fumio Yamauchi, Yuji Kamioka, Tetsuya Yano, et al.

Cancer Res Published OnlineFirst August 3, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-3534

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2016/08/03/0008-5472.CAN-15-3534.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2016/08/31/0008-5472.CAN-15-3534. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.