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) Ó2016 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 Ó2016 American Association for Cancer Research. approaches for the host cells, transgenic mice expressing the FRET www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst August 3, 2016; DOI: 10.1158/0008-5472.CAN-15-3534 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