Multimodal VEGF-Targeted Contrast-Enhanced Ultrasound and Photoacoustic Imaging of Rats with Inflammatory Arthritis: Using Dye-VEGF-Antibody-Loaded Microbubbles

Owing to the heavy health burdens from rheumatoid arthritis, a sensitive and objective imaging method is needed for early diagnosis and accurate evaluation of the disease. We aimed to fabricate vascular epithelial growth factor (VEGF)–targeted microbubbles (MBs) to evaluate the expression levels of VEGF within the inflammatory lesions of rats with adjuvant-induced arthritis (AIA) using a multimodal photoacoustic (PA)/ultrasound (US) imaging system. Fluorescein isothiocyanate–biotin double-labeled vascular endothelial growth factor receptor 2 antibodies and Cy5.5–biotin double-labeled VEGF2 antibodies were added to the avidin-labeled MBs to synthesize VEGF-targeted MBs. The antibodies could specifically bind to the MBs according to the flow cytometry and fluorescence imaging. In vitro experiments on the cellular uptake of the target MBs also validated the interaction of the VEGF antibodies and the MBs. Multimodal contrast-enhanced US (CEUS)/PA imaging was performed in sequence on the inflamed paws of the AIA rats with a single PA/US imaging system after the injection of the targeted MBs. The CEUS and PA signals were then quantified and verified by the pathologic results. A CEUS pattern of fast wash in and slow washout was observed in the AIA rats after injection of targeted MBs. Compared with AIA rats injected with unconnected VEGF antibodies and naked MBs, AIA rats injected with targeted MBs presented a higher peak intensity (p = 0.0079 and 0.0079 respectively) and a longer time to peak (p = 0.0117 and 0.0117, respectively). The PA signals were also significantly enhanced after injection of targeted MBs (p = 0.0112 and 0.0119, respectively), which was in accordance with the pathologic and immunohistochemical results. In conclusion, VEGF-targeted MBs can be used as agents for multimodal CEUS/PA imaging and to detect VEGF expression in the inflammatory lesions of AIA rats in vivo. This strategy may be useful in imaging evaluation of arthritis by identifying inflammation-related molecules in different imaging modes.     

Time-course imaging of therapeutic functional tumor vascular normalization by antiangiogenic agents

We describe here new technology that enables noninvasive imaging of therapeutic functional normalization of tumor blood vessels by antiangiogenic agents. Noninvasive variable-magnification in vivo-fluorescence imaging as well as fluorescence tomography was used to visualize functional vessel normalization. Changes in the same vessel before and after drug treatment were imaged with high resolution in real time. Differences in vascular responses to the mTOR inhibitor rapamycin and to an anti-VEGF antibody were functionally imaged. Tumor vessel normalization was shown by significantly reduced leakiness and subsequent improved tumor delivery of Paclitaxel-BODPY as well as by normalized morphology. The tumor vascular pool agent, AngioSense(750), was retained only in tumors after either anti-VEGF antibody or rapamycin treatment, as visualized by noninvasive fluorescence tomography. The antiangiogenic therapy normalized vessels, which significantly enhanced the antitumor efficacy of paclitaxel because of increased drug penetration throughout the tumor. The optical imaging technology described here is thus a powerful, noninvasive, time-course imaging tool of functional tumor vessel normalization and its therapeutic consequences.     

Aqueous proteins help predict the response of patients with neovascular age-related macular degeneration to anti-VEGF therapy

BackgroundTo reduce the treatment burden for patients with neovascular age-related macular degeneration (nvAMD), emerging therapies targeting vascular endothelial growth factor (VEGF) are being designed to extend the interval between treatments, thereby minimizing the number of intraocular injections. However, which patients will benefit from longer-acting agents is not clear.MethodsEyes with nvAMD (n = 122) underwent 3 consecutive monthly injections with currently available anti-VEGF therapies, followed by a treat-and-extend protocol. Patients who remained quiescent 12 weeks from their prior treatment entered a treatment pause and were switched to pro re nata (PRN) treatment (based on vision, clinical exam, and/or imaging studies). Proteomic analysis was performed on aqueous fluid to identify proteins that correlate with patients’ response to treatment.ResultsAt the end of 1 year, 38 of 122 eyes (31%) entered a treatment pause (≥30 weeks). Conversely, 21 of 122 eyes (17%) failed extension and required monthly treatment at the end of year 1. Proteomic analysis of aqueous fluid identified proteins that correlated with patients’ response to treatment, including proteins previously implicated in AMD pathogenesis. Interestingly, apolipoprotein-B100 (ApoB100), a principal component of drusen implicated in the progression of nonneovascular AMD, was increased in treated patients who required less frequent injections. ApoB100 expression was higher in AMD eyes compared with controls but was lower in eyes that develop choroidal neovascularization (CNV), consistent with a protective role. Accordingly, mice overexpressing ApoB100 were partially protected from laser-induced CNV.FundingThis work was supported by the National Eye Institute, National Institutes of Health grants R01EY029750, R01EY025705, and R01 EY27961; the Research to Prevent Blindness, Inc.; the Alcon Research Institute; and Johns Hopkins University through the Robert Bond Welch and Branna and Irving Sisenwein professorships in ophthalmology.ConclusionAqueous biomarkers could help identify patients with nvAMD who may not require or benefit from long-term treatment with anti-VEGF therapy.     

Spatial light modulator based active wide-field illumination for ex vivo and in vivo quantitative NIR FRET imaging

Fluorescence lifetime imaging is playing an increasing role in drug development by providing a sensitive method to monitor drug delivery and receptor-ligand interactions. However, the wide dynamic range of fluorescence intensity emitted by ex vivo and in vivo samples presents challenges in retrieving information over the whole subject accurately and quantitatively. To overcome this challenge, we developed an active wide-field illumination (AWFI) strategy based on a spatial light modulator that acquires optimal fluorescence signals by enhancing the dynamic range, signal to noise ratio, and estimation of lifetime-based parameters. We demonstrate the ability of AWFI to estimate Förster resonance energy transfer (FRET) donor fraction from dissected organs with high accuracy (standard deviation <6%) over the whole field of view, in contrast with the homogenous wide-field illumination. We further report its successful application to quantitative FRET imaging in a live mouse. AWFI allows improved detection of weak signals and enhanced quantitative accuracy in ex vivo and in vivo molecular fluorescence quantitative imaging. The technique allows for robust quantitative estimation of the bio-distribution of molecular probes and lifetime-based parameters over an extended imaging field exhibiting a large range of fluorescence intensities and at a high acquisition speed (less than 1 min).OCIS codes: (260.2510) Fluorescence, (170.6920) Time-resolved imaging, (170.3650) Lifetime-based sensing, (170.2945) Illumination design, (230.6120) Spatial light modulators, (260.2160) Energy transfer     

Sustained delivery of anti-VEGF from injectable hydrogel systems provides a prolonged decrease of endothelial cell proliferation and angiogenesis in vitro

Therapeutic antibodies are attractive treatment options for numerous diseases based on their ability to target and bind to specific proteins or antigens. Bevacizumab, an antiangiogenic antibody, has shown promise for multiple diseases, including various cancers and macular degeneration, where excessive VEGF secretion induces aberrant angiogenesis. In many cases local, sustained delivery of a therapeutic antibody would be preferable to maximize the therapeutic at the disease site, eliminate the need for repeated doses, and reduce systemic side effects. The biodegradable polysaccharides alginate and chitosan can electrostatically interact to form a polyelectrolyte complex (PEC), and have proved effective as a carrier for controlled release of antibodies. In this work, an alginate–chitosan PEC system was designed to produce targeted 30-day delivery of non-specific IgG and anti-VEGF antibodies. The release of anti-VEGF was slow relative to IgG release, suggesting that release rate is antibody specific and is based on the interactions of the PEC with charges present on the antibody surface. The anti-VEGF released from the PEC was shown to successfully inhibit VEGF-induced proliferation and angiogenesis in vitro throughout the 30-day test period.

Sustained delivery of bioactive anti-VEGF antibodies is demonstrated using a polyelectrolyte complex of alginate and chitosan. The released anti-VEGF inhibited VEGF induced-proliferation and angiogenesis in HUVECs over a 30-day period.     

K562细胞血管内皮生长因子自分泌链作用的实验研究

目的探讨抗VEGF抗体及抗VEGF发夹状核酶基因对K562细胞增殖、凋亡及相关基因表达的影响及其分子机制。方法将不同浓度的VEGF抗体作用于K562细胞，应用脂质体介导的方法将抗VEGF发夹状核酶基因真核表达载体pcDNA-RZ转染K562细胞，采用MTT法、甲基纤维素半固体培养法和细胞周期测定分析抗VEGF抗体及发夹状核酶基因对白血病细胞增殖的影响；DNA凝胶电泳和AnnexinⅤ标记法检测白血病细胞的凋亡程度；RT-PCR法测定K562细胞中相关基因表达的变化。结果抗VEGF抗体能抑制K562细胞生长，促进其凋亡，这一作用呈剂量依赖关系。0．165μg／ml VEGF抗体作用于K562细胞72h，DNA凝胶电泳出现梯状条带，当抗体浓度增加到0．825μg／ml时，梯状条带更为清晰；RT-PCR显示，VEGF抗体作用后，K562细胞MRP、TOPOⅡ基因的表达较对照组下调，而GST基因表达无明显改变。与K562及K562／PC细胞（转染空质粒的K562细胞）相比，转染抗VEGF核酶基因的K562／RZ细胞VEGF mRNA和蛋白的表达量明显降低；生长曲线提示K562／RZ细胞生长缓慢，甲基纤维素集落形成率较对照组明显下降，对照组集落形成率为（30．5±3．3）％，而K562／RZ组为（16．3±2．8）％，细胞周期动力学分析结果显示K562／RZ细胞G1期细胞增多，S期细胞显著减少；在小剂量As2O3作用下，K562／RZ细胞凋亡数量较对照明显增高（凋亡率对照组为13．4％，K562／RZ组为31．5％）。结论抗VEGF抗体阻断K562细胞VEGF自分泌环路或者抗VEGF发夹状核酶减少K562细胞中VEGF的合成，能抑制K562细胞的增殖，促进K562细胞的凋亡，引起部分与细胞凋亡和多药耐药相关的基因表达改变。     

结直肠癌螺旋CT灌注成像与肿瘤血管生成的相关性

目的探讨结、直肠癌螺旋CT(SCT)灌注成像与其血管生成的相关性。方法对30例结、直肠癌患者进行SCT单层动态增强扫描,绘制所选层面的癌灶、靶动脉的感兴趣区的时间-密度曲线(TDC)。根据TDC计算癌灶的血流灌注量(PF)。测定癌组织的微血管密度(MVD)和血管内皮生长因子(VEGF)的表达。将结、直肠癌的PF与MVD、VEGF的表达进行比较,且三者亦分别与临床病理因素(Dukes分期、浆膜浸润及淋巴结转移)进行比较。结果结、直肠癌的PF随着病理分期期别的升高有下降趋势,与MVD的趋势一致。VEGF的表达阳性率为87%(26/30)。PF与MVD、VEGF的表达之间均无显著相关性(r分别为0.16,0.19,P值均>0.05)。MVD与VEGF间也无显著相关性(r=-0.03,P>0.05);PF、MVD及VEGF在Dukes分期、浆膜浸润、淋巴结转移比较中差异均无统计学意义。结论SCT灌注成像与MVD、VEGF均能反映结、直肠癌的血管生成状况,但它们之间无明显相关性。     

Shortwave-infrared (SWIR) fluorescence molecular imaging using indocyanine green–antibody conjugates for the optical diagnostics of cancerous tumours

Recently, shortwave-infrared (SWIR, 1000–1400 nm) fluorescence imaging has attracted much attention due to the higher contrast and sensitivity with deeper penetration depths compared to conventional visible and near-infrared (NIR) fluorescence imaging. For the SWIR fluorescence imaging, the development of fluorescent probes emitting over 1000 nm is necessary. So far, a variety of SWIR fluorescent probes based on single-walled carbon nanotubes, quantum dots, rare-metal doped nanomaterials, and organic dyes have been developed. However, there are a very limited number of biocompatible SWIR fluorescent probes, which can be used to biomedical applications. Among NIR and SWIR fluorescent probes, indocyanine green (ICG) is the only fluorescent dye approved by US Food and Drug Administration (FDA) for clinical use. Although ICG has a fluorescence maximum at a NIR region (ca. 830 nm), ICG emits in the SWIR region over 1000 nm. Here, we present ICG-based SWIR fluorescence molecular imaging for the highly-sensitive optical detection of breast and skin tumours in mice. As SWIR fluorescent molecular-imaging probes, we synthesized ICG–antibody conjugates, which prepared from anti-HER2 antibody (Herceptin), anti-EGFR antibody (Erbitux), anti-VEGFR-2 antibody (Cyramza), and anti-PD-L1 antibody (anti-PD-L1 ab). The present SWIR molecular imaging probes specifically accumulated to the breast and skin tumours, and their SWIR fluorescence images (>1000 nm) showed 1.5–2.0 times higher contrast than NIR tumour images taken at 830 nm. We show that the SWIR fluorescence imaging using ICG–antibody conjugates can be used for the elucidation of expression level of cancer-specific membrane proteins, HER2, EGFR, VEGFR-2, and PD-L1 in vivo. We also show that the SWIR fluorescence imaging enables quantitative analysis of the change in the size of tumour treated with an anti-cancer drug, Kadcyla. Our findings suggest that the SWIR fluorescence molecular imaging using ICG–antibody conjugates has potential to use for the optical diagnostics of cancerous tumors in medical and clinical fields.

We present indocyanine green (ICG)-based shortwave-infrared (SWIR) fluorescence molecular imaging for the highly-sensitive optical detection of breast and skin tumours in mice.     

Contrast ultrasound imaging for identification of early responder tumor models to anti-angiogenic therapy

Agents targeting vascular endothelial growth factor (VEGF) have been validated as cancer therapeutics, yet efficacy can differ widely between tumor types and individual patients. In addition, such agents are costly and can have significant toxicities. Rapid noninvasive determination of response could provide significant benefits. We tested if response to the anti-VEGF antibody bevacizumab (BV) could be detected using contrast-enhanced ultrasound imaging (CEUS). We used two xenograft model systems with previously well-characterized responses to VEGF inhibition, a responder (SK-NEP-1) and a non-responder (NGP), and examined perfusion-related parameters. CEUS demonstrated that BV treatment arrested the increase in blood volume in the SK-NEP-1 tumor group only. Molecular imaging of α(V)β(3) with targeted microbubbles was a more sensitive prognostic indicator of BV efficacy. CEUS using RGD-labeled microbubbles showed a robust decrease in α(V)β(3) vasculature following BV treatment in SK-NEP-1 tumors. Paralleling these findings, lectin perfusion assays detected a disproportionate pruning of smaller, branch vessels. Therefore, we conclude that the response to BV can be identified soon after initiation of treatment, often within 3 days, by use of CEUS molecular imaging techniques. The use of a noninvasive ultrasound approach may allow for earlier and more effective determination of efficacy of antiangiogenic therapy.     

人前列腺癌靶向超声造影剂对荷瘤裸鼠靶向显像的研究

目的：探讨携前列腺特异性膜抗原（PSMA）抗体和携血管内皮生长因子（VEGF）抗体的靶向超声造影剂对荷人前列腺癌裸鼠的特异增强显像效果。方法：人前列腺癌细胞株LNCaP体外无菌条件下培养传代，BALB／c-nu雄性裸鼠皮下接种，建立荷人前列腺癌裸鼠动物模型。分别经荷瘤裸鼠尾静脉推注普通造影剂和携PSMA抗体及携VEGF抗体的靶向造影剂12min后采用能量多普勒成像，测量并计算肿瘤截面内血流信号所占的面积比，并分析比较普通造影剂和靶向造影剂对前列腺癌组织能量多普勒信号的增强效果。结果：成功建立了荷人前列腺癌裸鼠动物模型，推注携带VEGF抗体的靶向造影剂超声造影后行能量多普勒显像，可见前列腺癌组织能量多普勒信号显著增强，与普通造影剂和携带PSMA抗体的靶向造影剂相比较，差异有统计学意义（P〈0．01）。结论：VEGF抗体介导的靶向超声造影剂在体内可以有效特异性地与前列腺肿瘤新生血管内皮细胞结合，可作为诊断前列腺癌的靶向造影剂。能量多普勒延迟显像能有效显示靶向造影后肿瘤组织内的能量多普勒增强信号。     

Inhibition of Choroidal Neovascularization in a Nonhuman Primate Model by Intravitreal Administration of an AAV2 Vector Expressing a Novel Anti-VEGF Molecule

Inhibition of vascular endothelial growth factor (VEGF) for the management of the pathological ocular neovascularization associated with diseases such as neovascular age-related macular degeneration is a proven paradigm; however, monthly intravitreal injections are required for optimal treatment. We have previously shown that a novel, secreted anti-VEGF molecule sFLT01 delivered by intravitreal injection of an AAV2 vector (AAV2-sFLT01) gives persistent expression and is efficacious in a murine model of retinal neovascularization. In the present study, we investigate transduction and efficacy of an intravitreally administered AAV2-sFLT01 in a nonhuman primate (NHP) model of choroidal neovascularization (CNV). A dose-dependent and persistent expression of sFLT01 was observed by collecting samples of aqueous humor at different time points over 5 months. The location of transduction as elucidated by in situ hybridization was in the transitional epithelial cells of the pars plana and in retinal ganglion cells. AAV2-sFLT01 was able to effectively inhibit laser-induced CNV in a dose-dependent manner as determined by comparing the number of leaking CNV lesions in the treated versus control eyes using fluorescein angiography. Our data suggest that intravitreal delivery of AAV2-sFLT01 may be an effective long-term treatment for diseases caused by ocular neovascularization.     

Assessment of Molecular Acoustic Angiography for Combined Microvascular and Molecular Imaging in Preclinical Tumor Models

Purpose

The purposes of the present study is to evaluate a new ultrasound molecular imaging approach in its ability to image a preclinical tumor model and to investigate the capacity to visualize and quantify co-registered microvascular and molecular imaging volumes.

Procedures

Molecular imaging using the new technique was compared with a conventional ultrasound molecular imaging technique (multi-pulse imaging) by varying the injected microbubble dose and scanning each animal using both techniques. Each of the 14 animals was randomly assigned one of three doses; bolus dose was varied, and the animals were imaged for three consecutive days so that each animal received every dose. A microvascular scan was also acquired for each animal by administering an infusion of nontargeted microbubbles. These scans were paired with co-registered molecular images (VEGFR2-targeted microbubbles), the vessels were segmented, and the spatial relationships between vessels and VEGFR2 targeting locations were analyzed. In five animals, an additional scan was performed in which the animal received a bolus of microbubbles targeted to E- and P-selectins. Vessel tortuosity as a function of distance from VEGF and selectin targeting was analyzed in these animals.

Results

Although resulting differences in image intensity due to varying microbubble dose were not significant between the two lowest doses, superharmonic imaging had significantly higher contrast-to-tissue ratio (CTR) than multi-pulse imaging (mean across all doses 13.98 dB for molecular acoustic angiography vs. 0.53 dB for multi-pulse imaging; p = 4.9 × 10^?10). Analysis of registered microvascular and molecular imaging volumes indicated that vessel tortuosity decreases with increasing distance from both VEGFR2- and selectin-targeting sites.

Conclusions

Molecular acoustic angiography (superharmonic molecular imaging) exhibited a significant increase in CTR at all doses tested due to superior rejection of tissue artifact signals. Due to the high resolution of acoustic angiography molecular imaging, it is possible to analyze spatial relationships in aligned microvascular and molecular superharmonic imaging volumes. Future studies are required to separate the effects of biomarker expression and blood flow kinetics in comparing local tortuosity differences between different endothelial markers such as VEGFR2, E-selectin, and P-selectin.

     

实时荧光定量RT-PCR检测乳腺癌VEGF mRNA的表达

目的：建立实时荧光定量逆转录一多聚酶链反应（real—timequantitative,RT—PCR）检测乳腺癌患者肿瘤组织VEGFmRNA表达的方法,并验证其与免疫组织化学LsAB方法检测VEGF蛋白表达的相关性。方法：提取组织总RNA,逆转录成mRNA,采用实时荧光定量RT—PCR检测44例乳腺浸润性导管癌、25例癌旁正常小叶组织、13例乳腺良性疾病组织中VEGFmRNA表达;采用免疫组织化学LSAB法和彩色图像病理分析系统半定量检测其VEGF蛋白表达。结果：乳腺癌组织中VEGFmRNA表达水平显著高于癌旁和良性病组织（｜P〈0．001）,而后两者之间无明显差异（P〉0．05）;VEGF蛋白表达在癌组织,癌旁和良性病组中的分布特点与VEGFmRNA完全一致。结论;本研究所建立的用于检测人乳腺组织VEGFmRNA表达的实时荧光定量RT—PCR方法,是一种快速有效、灵敏度高、特异性好的定量检测方法,与免疫组化LSAB方法检测的VEGF蛋白表达具有良好相关性,在乳腺癌的早期诊断和良恶性鉴别诊断中具有重要的参考价值。     

Fast microbubble dwell-time based ultrasonic molecular imaging approach for quantification and monitoring of angiogenesis in cancer

PURPOSE: To develop and test a fast ultrasonic molecular imaging technique for quantification and monitoring of angiogenesis in cancer. MATERIALS AND METHODS: A new software algorithm measuring the dwell time of contrast microbubbles in near real-time (henceforth, fast method) was developed and integrated in a clinical ultrasound system. In vivo quantification and monitoring of tumor angiogenesis during anti-VEGF antibody therapy was performed in human colon cancer xenografts in mice (n=20) using the new fast method following administration of vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast microbubbles. Imaging results were compared with a traditional destruction/replenishment approach (henceforth, traditional method) in an intra-animal comparison. RESULTS: There was excellent correlation (R(2)=0.93; P<0.001) between the fast method and the traditional method in terms of VEGFR2-targeted in vivo ultrasonic molecular imaging with significantly higher (P=0.002) imaging signal in colon cancer xenografts using VEGFR2-targeted compared to control non-targeted contrast microbubbles. The new fast method was highly reproducible (ICC=0.87). Following anti-angiogenic therapy, ultrasonic molecular imaging signal decreased by an average of 41±10%, whereas imaging signal increased by an average of 54±8% in non-treated tumors over a 72-hour period. Decreased VEGFR2 expression levels following anti-VEGF therapy were confirmed on ex vivo immunofluorescent staining. CONCLUSIONS: Fast ultrasonic molecular imaging based on dwell time microbubble signal measurements correlates well with the traditional measurement method, and allows reliable in vivo monitoring of anti-angiogenic therapy in human colon cancer xenografts. The improved work-flow afforded by the new quantification approach may facilitate clinical translation of ultrasonic molecular imaging.     

Effects of anti-VEGF on pharmacokinetics, biodistribution, and tumor penetration of trastuzumab in a preclinical breast cancer model

Both human epidermal growth factor receptor 2 (HER-2/neu) and VEGF overexpression correlate with aggressive phenotypes and decreased survival among breast cancer patients. Concordantly, the combination of trastuzumab (anti-HER2) with bevacizumab (anti-VEGF) has shown promising results in preclinical xenograft studies and in clinical trials. However, despite the known antiangiogenic mechanism of anti-VEGF antibodies, relatively little is known about their effects on the pharmacokinetics and tissue distribution of other antibodies. This study aimed to measure the disposition properties, with a particular emphasis on tumor uptake, of trastuzumab in the presence or absence of anti-VEGF. Radiolabeled trastuzumab was administered alone or in combination with an anti-VEGF antibody to mice bearing HER2-expressing KPL-4 breast cancer xenografts. Biodistribution, autoradiography, and single-photon emission computed tomography-X-ray computed tomography imaging all showed that anti-VEGF administration reduced accumulation of trastuzumab in tumors despite comparable blood exposures and similar distributions in most other tissues. A similar trend was also observed for an isotype-matched IgG with no affinity for HER2, showing reduced vascular permeability to macromolecules. Reduced tumor blood flow (P < 0.05) was observed following anti-VEGF treatment, with no significant differences in the other physiologic parameters measured despite immunohistochemical evidence of reduced vascular density. In conclusion, anti-VEGF preadministration decreased tumor uptake of trastuzumab, and this phenomenon was mechanistically attributed to reduced vascular permeability and blood perfusion. These findings may ultimately help inform dosing strategies to achieve improved clinical outcomes.     

Feasibility,sensitivity, and reliability of laser-induced fluorescence imaging of green fluorescent protein-expressing tumors in vivo

Whole-body imaging of green fluorescent protein (GFP) can be used to test the efficiency of gene carriers for in vivo transduction. The aim of the current study was to determine the sensitivity and the accuracy of a GFP imaging procedure by in vivo investigation of GFP-expressing tumor cells. An improved method of whole-body GFP imaging made use of a laser excitation source and band-pass filters matched specifically to GFP and constitutive tissue fluorescence emission bands. Processing of the primary GFP fluorescence images acquired by the CCD camera subtracted background tissue autofluorescence. Our approach achieved 100% sensitivity and specificity for in vivo detection of 10%-transfected BxPc3 pancreatic tumor after subcutaneous grafting or orthotopical implantation in the pancreas of nude mice. It also detected less transfected tumors (i.e., 1 to 5%) but with a loss in sensitivity (50% of cases). The system was employed over a 5-week period to monitor the persistence of GFP expression in 10%-transfected BxPc3 tumors orthotopically implanted in the pancreas of two nude mice, allowing the direct visualization of tumor progression and spread. In facilitating the temporal–spatial follow-up of GFP expression in vivo, the optimized laser-induced fluorescence imaging device can support preclinical investigations of vectors for therapeutic gene transduction through regular, harmless, real-time monitoring of theirin vivo transductional efficacy and persistence.     

Inhibitory effects of antivascular endothelial growth factor strategies in experimental dopamine-resistant prolactinomas

Prolactin-secreting adenomas are the most frequent type among pituitary tumors, and pharmacological therapy with dopamine agonists remains the mainstay of treatment. But some adenomas are resistant, and a decrease in the number or function of dopamine D2 receptors (D2Rs) has been described in these cases. D2R knockout [Drd2(-/-)] mice have chronic hyperprolactinemia and pituitary hyperplasia and provide an experimental model for dopamine agonist-resistant prolactinomas. We described previously that disruption of D2Rs increases vascular endothelial growth factor (VEGF) expression. We therefore designed two strategies of antiangiogenesis using prolactinomas generated in Drd2(-/-) female mice: direct intra-adenoma mVEGF R1 (Flt-1)/Fc chimera (VEGF-TRAP) injection for 3 weeks [into subcutaneously transplanted pituitaries from Drd2(-/-) mice] and systemic VEGF neutralization with the specific monoclonal antibody G6-31. Both strategies resulted in substantial decrease of prolactin content and lactotrope area, and a reduction in tumor size was observed in in situ prolactinomas. There were significant decreases in vascularity, evaluated by cluster of differentiation molecule 31 vessel staining, and proliferation (proliferating cell nuclear antigen staining) in response to both anti-VEGF treatments. These data demonstrate that the antiangiogenic approach was effective in inhibiting the growth of in situ dopamine-resistant prolactinomas as well as in the transplanted adenomas. No differences in VEGF protein expression were observed after either anti-VEGF treatment, and, although serum VEGF was increased in G6-31-treated mice, pituitary activation of the VEGF receptor 2 signaling pathway was reduced. Our results indicate that, even though the role of angiogenesis in pituitary adenomas is contentious, VEGF might contribute to adequate vascular supply and represent a supplementary therapeutic target in dopamine agonist-resistant prolactinomas.     

In vivo fluorescence lifetime tomography of a FRET probe expressed in mouse

Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct.OCIS codes: (170.2655) Functional monitoring and imaging, (170.3010) Image reconstruction techniques, (170.3650) Lifetime-based sensing, (170.3660) Light propagation in tissues, (170.3880) Medical and biological imaging, (170.6960) Tomography     

Anti-VEGF therapy: the search for clinical biomarkers

Despite the large diffusion and rapid development of anti-VEGF therapy in clinical practice and in contrast to the consolidated evidence with imatinib and trastuzumab that demonstrated a direct correlation between pre-treatment target expression and drug activity, it is very difficult, at present, to identify validated and useful biomarkers to monitor the efficacy of these compounds and to appropriately select patients most likely to benefit from such treatments. However, emerging data suggest that this is not presently feasible for antiangiogenic drugs. Although tumoral and/or circulating VEGF levels have been associated with tumor progression and/or poor prognosis, to date, there is no validated evidence suggesting their role as potential predictive biomarkers of response to anti-VEGF therapy. Recently, many studies have documented promising results with the evaluation of circulating endothelial cells and/or progenitors, and the use of several imaging techniques, such as dynamic contrast-enhanced MRI, PET, dynamic CT scan and functional ultrasound. These preliminary data need a validation in larger prospective trials.