Direct site-specific labeling of the Cys-tag moiety in scVEGF with technetium 99m

Angiogenesis is a fundamental feature of tumor development, and therefore, the tracers for molecular imaging of specific angiogenic biomarkers are expected to be useful for diagnostics, patient monitoring, and drug development. We have created a new class of imaging agents based on the most important mediator of angiogenesis, vascular endothelial growth factor (VEGF). Our latest version is a single-chain (sc) VEGF protein containing an N-terminal Cys-tag designed for site-specific modification with a variety of imaging and therapeutic moieties. We have recently found that the Cys-tag itself can form a stable chelate with (99m)Tc using tin-tricine as an exchange reagent. This self-chelation approach yields a highly stable and fully functional form of radiolabeled scVEGF that can be used as a SPECT tracer for tumor angiogenesis. Also of note is that directly labeled scVEGF has less than one-half the nonspecific renal uptake of (99m)Tc-HYNIC-scVEGF. The simple production of scVEGF for direct chelation of (99m)Tc makes it a promising molecular imaging agent for the oncology clinic.     

Molecular MRI assessment of vascular endothelial growth factor receptor‐2 in rat C6 gliomas

Angiogenesis is essential to tumour progression and a precise evaluation of angiogenesis is important for tumour early diagnosis and treatment. The quantitative and dynamic in vivo assessment of tumour angiogenesis can be achieved by molecular magnetic resonance imaging (mMRI). Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are the main regulatory systems in angiogenesis and have been used as hot targets for radionuclide‐based molecular imaging. However, little research has been accomplished in targeting VEGF/VEGFRs by mMRI. In our study, we aimed to assess the expression of VEGFR2 in C6 gliomas by using a specific molecular probe with mMRI. The differential uptake of the probe conjugated to anti‐VEGFR2 monoclonal antibody, shown by varied increases in T₁ signal intensity during a 2 hr period, demonstrated the heterogeneous expression of VEGFR2 in different tumour regions. Microscopic fluorescence imaging, obtained for the biotin group in the probe with streptavidin‐Cy3, along with staining for cellular VEGFR2 levels, laminin and CD45, confirmed the differential distribution of the probe which targeted VEGFR2 on endothelial cells. The angiogenesis process was also assessed using magnetic resonance angiography, which quantified tumour blood volume and provided a macroscopic view and a dynamic change of the correlation between tumour vasculature and VEGFR2 expression. Together these results suggest mMRI can be very useful in assessing and characterizing the expression of specific angiogenic markers in vivo and help evaluate angiogenesis associated with tumour progression.     

Concomitant vascular GRP-receptor and VEGF-receptor expression in human tumors: molecular basis for dual targeting of tumoral vasculature

Gastrin-releasing peptide (GRP) and GRP receptors (GRPR) play a role in tumor angiogenesis. Recently, GRPR were found to be frequently expressed in the vasculature of a large variety of human cancers. Here, we characterize these GRPR by comparing the vascular GRPR expression and localization in a selection of human cancers with that of an established biological marker of neoangiogenesis, the vascular endothelial growth factor (VEGF) receptor. In vitro quantitative receptor autoradiography was performed in parallel for GRPR and VEGF receptors (VEGFR) in 32 human tumors of various origins, using ¹²⁵I-Tyr-bombesin and ¹²⁵I-VEGF₁₆₅ as radioligands, respectively. Moreover, VEGFR-2 was evaluated immunohistochemically. All tumors expressed GRPR and VEGFR in their vascular system. VEGFR were expressed in the endothelium in the majority of the vessels. GRPR were expressed in a subpopulation of vessels, preferably in their muscular coat. The vessels expressing GRPR were all VEGFR-positive whereas the VEGFR-expressing vessels were not all GRPR-positive. GRPR expressing vessels were found immunohistochemically to co-express VEGFR-2. Remarkably, the density of vascular GRPR was much higher than that of VEGFR. The concomitant expression of GRPR with VEGFR appears to be a frequent phenomenon in many human cancers. The GRPR, localized and expressed in extremely high density in a subgroup of vessels, may function as target for antiangiogenic tumor therapy or angiodestructive targeted radiotherapy with radiolabeled bombesin analogs alone, or preferably together with VEGFR targeted therapy.     

CD34+VEGFR‐3+ progenitor cells have a potential to differentiate towards lymphatic endothelial cells

Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization. However, it is poorly understood whether EPCs contribute to lymphangiogenesis. Here, we assessed differentiation of a novel population of EPCs towards lymphatic endothelial cells and their lymphatic formation. CD34⁺VEGFR‐3⁺ EPCs were isolated from mononuclear cells of human cord blood by fluorescence‐activated cell sorting. These cells expressed CD133 and displayed the phenotype of the endothelial cells. Cell colonies appeared at 7–10 days after incubation. The cells of the colonies grew rapidly and could be repeatedly subcultured. After induction with VEGF‐C for 2 weeks, CD34⁺VEGFR‐3⁺ EPCs could differentiate into lymphatic endothelial cells expressing specific markers 5′‐nucleotidase, LYVE‐1 and Prox‐1. The cells also expressed hyaluronan receptor CD44. The differentiated cells had properties of proliferation, migration and formation of lymphatic capillary‐like structures in three‐dimensional collagen gel and Matrigel. VEGF‐C enhanced VEGFR‐3 mRNA expression. After interfering with VEGFR‐3 siRNA, the effects of VEGF‐C were diminished. These results demonstrate that there is a population of CD34⁺VEGFR‐3⁺ EPCs with lymphatic potential in human cord blood. VEGF‐C/VEGFR‐3 signalling pathway mediates differentiation of CD34⁺VEGFR‐3⁺ EPCs towards lymphatic endothelial cells and lymphangiogenesis. Cord blood‐derived CD34⁺VEGFR‐3⁺ EPCs may be a reliable source in transplantation therapy for lymphatic regenerative diseases.     

99mTc-bioorthogonal click chemistry reagent for in vivo pretargeted imaging

Metal-free click chemistry has become an important tool for pretargeted approaches in the molecular imaging field. The application of bioorthogonal click chemistry between a pretargeted trans-cyclooctene (TCO) derivatized monoclonal antibody (mAb) and a ^99mTc-modified 1,2,4,5-tetrazine for tumor imaging was examined in vitro and in vivo. The HYNIC tetrazine compound was synthesized and structurally characterized, confirming its identity. Radiolabeling studies demonstrated that the HYNIC tetrazine was labeled with ^99mTc at an efficiency of >95% and was radiochemically stable. ^99mTc–HYNIC tetrazine reacted with the TCO–CC49 mAb in vitro demonstrating its selective reactivity. In vivo biodistribution studies revealed non-specific liver and GI uptake due to the hydrophobic property of the compound, however pretargeted SPECT imaging studies demonstrated tumor visualization confirming the success of the cycloaddition reaction in vivo. These results demonstrated the potential of ^99mTc–HYNIC–tetrazine for tumor imaging with pretargeted mAbs.     

The synthesis of pteroyl-lys conjugates and its application as Technetium-99m labeled radiotracer for folate receptor-positive tumor targeting

Aiming to develop a new ^99mTc-labeled folate derivative for FR-positive tumor imaging, a simpler method has been established to synthesize the folate-drug conjugates with free α-carboxyl group. In this study, the conjugate pteroyl-lys-HYNIC was synthesized and labeled with ^99mTc using tricine and TPPTS as co-ligands. The radiochemical purity of the final complex ^99mTc(HYNIC-lys-pteroyl)(tricine/TPPTS), 5 was high (>98%), and it remained stable in saline and plasma over 6 h after preparation. The biologic evaluation results showed that the ^99mTc labeled pteroyl-lys conjugate was able to specifically target the FR-positive tumor cells and tissues both in vitro and in vivo, highlighting its potential as an effective folate receptor targeted agent for tumor imaging.     

A Novel 99mTc-Labeled Molecular Probe for Tumor Angiogenesis Imaging in Hepatoma Xenografts Model: A Pilot Study

Introduction

Visualization of tumor angiogenesis using radionuclide targeting provides important diagnostic information. In previous study, we proved that an arginine-arginine-leucine (RRL) peptide should be a tumor endothelial cell specific binding sequence. The overall aim of this study was to evaluate whether ^99mTc-radiolabeled RRL could be noninvasively used for imaging of malignant tumors in vivo, and act as a new molecular probe targeting tumor angiogenesis.

Methods

The RRL peptide was designed and radiosynthesized with ^99mTc by a one-step method. The radiolabeling efficiency and radiochemical purity were then characterized in vitro. ^99mTc-RRL was injected intravenously in HepG2 xenograft-bearing BALB/c nude mice. Biodistribution and in vivo imaging were performed periodically. The relationship between tumor size and %ID uptake of ^99mTc-RRL was also explored.

Results

The labeling efficiencies of ^99mTc-RRL reached 76.9%±4.5% (n = 6) within 30–60 min at room temperature, and the radiochemical purity exceeded 96% after purification. In vitro stability experiment revealed the radiolabeled peptide was stable. Biodistribution data showed that ^99mTc-RRL rapidly cleared from the blood and predominantly accumulated in the kidneys and tumor. The specific uptake of ^99mTc-RRL in tumor was significantly higher than that of unlabeled RRL blocking and free pertechnetate control test after injection (p<0.05). The ratio of the tumor-to-muscle exceeded 6.5, tumor-to-liver reached 1.98 and tumor-to-blood reached 1.95. In planar gamma imaging study, the tumors were imaged clearly at 2–6 h after injection of ^99mTc-RRL, whereas the tumor was not imaged clearly in blocking group. The tumor-to-muscle ratio of images with ^99mTc-RRL was comparable with that of ¹⁸F-FDG PET images. Immunohistochemical analysis verified the excessive vasculature of tumor. There was a linear relationship between the tumor size and uptake of ^99mTc-RRL with R² = 0.821.

Conclusion

^99mTc-RRL can be used as a potential candidate for visualization of tumor angiogenesis in malignant carcinomas.     

Synthesis and evaluation of 99mTc–2-[(3-carboxy-1-oxopropyl)amino]-2-deoxy-d-glucose as a potential tumor imaging agent

The 2-[(3-carboxy-1-oxopropyl)amino]-2-deoxy-d-glucose (CPADG) was synthesized and radiolabeled with ^99mTcO₄⁻ to obtain the ^99mTc–CPADG complex in high yield. It was stable over 6 h in saline at room temperature and in serum at 37 °C. The partition coefficient and electrophoresis results indicated that the complex was hydrophilic and cationic. In vitro cell studies showed there was an increase in the uptake of ^99mTc–CPADG as a function of incubation time and ^99mTc–CPADG was possibly transported via the glucose transporters. The biodistribution of ^99mTc–CPADG in mice bearing S 180 tumor showed that the complex accumulated in the tumor with high uptake and good retention. The tumor/blood and tumor/muscle ratios increased with time and reached 1.91 and 5.05 at 4 h post-injection. Single photon emission computed tomography (SPECT) image studies showed there was an obvious accumulation in tumor sites, suggesting ^99mTc–CPADG would be a promising candidate for tumor imaging.     

Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to VEGF receptor 2 (VEGFR2) on endothelial cells (ECs). Downstream activation of the extracellular related kinases 1/2 (ERK1/2) is important for angiogenesis to proceed. Receptor internalization has been implicated in VEGFR2 signaling, but its role in the activation of ERK1/2 is unclear. To explore this question we utilized pitstop and dynasore, two small molecule inhibitors of endocytosis. First, we confirmed that both inhibitors block the internalization of VEGFR2 in ECs. We then stimulated ECs with VEGF in the presence and absence of the inhibitors and examined VEGFR2 signaling to ERK1/2. Activation of VEGFR2 and C-Raf still occurred in the presence of the inhibitors, whereas the activation of MEK1/2 and ERK1/2 was abrogated. Therefore, although internalization is not required for activation of either VEGFR2 or C-Raf in ECs stimulated with VEGF, internalization is necessary to activate the more distal kinases in the cascade. Importantly, inhibition of internalization also prevented activation of ERK1/2 when ECs were stimulated with other pro-angiogenic growth factors, namely fibroblast growth factor 2 and hepatocyte growth factor. In contrast, the same inhibitors did not block ERK1/2 activation in fibroblasts or cancer cells stimulated with growth factors. Finally, we show that these small molecule inhibitors of endocytosis block angiogenesis in vitro and in vivo. Therefore, receptor internalization may be a generic requirement for pro-angiogenic growth factors to activate ERK1/2 signaling in human ECs, and targeting receptor trafficking may present a therapeutic opportunity to block tumor angiogenesis.     

Synthesis of Tc-99m labeled glucosamino-Asp-cyclic(Arg-Gly-Asp-d-Phe-Lys) as a potential angiogenesis imaging agent

Angiogenesis imaging agents for single photon emission computed tomography (SPECT) play a role in diagnosing tumor-induced angiogenesis as well as tumor metastasis. We synthesized and evaluated radiolabeled RGD glycopeptides by incorporation of the [^99mTc(CO)₃(H₂O)₃]⁺. ^99mTc labeled glucosamino-D-c(RGDfK) ([^99mTc]2) was prepared in 90–93% radiochemical yields (decay corrected). In vitro cell binding assays demonstrated selective binding [^99mTc]2 to human umbilical vein endothelial (HUVE) cells, with inhibition of binding to 37.3% of control levels by 10 μM of cold authentic compounds. In addition, [^99mTc]2 was shown to have high binding affinity to purified α_vβ₃ integrin (IC₅₀ = 1.5 nM). These results suggest that these radiolabeled RGD glycopeptides may have value for non-invasive assessment of angiogenesis.     

99mTc-labeled monomeric and dimeric NGR peptides for SPECT imaging of CD13 receptor in tumor-bearing mice

CD13 receptor plays a critical role in tumor angiogenesis and metastasis. We therefore aimed to develop ^99mTc-labeled monomeric and dimeric NGR-containing peptides, namely, NGR1 and NGR2, for SPECT imaging of CD13 expression in HepG2 hepatoma xenografts. Both NGR-containing monomer and dimer were synthesized and labeled with ^99mTc. In vivo receptor specificity was demonstrated by successful blocking of tumor uptake of ^99mTc-NGR dimer in the presence of 20 mg/kg NGR2 peptide. Western blot and immunofluorescence staining confirmed the CD13 expression in HepG2 cells. The NGR dimer showed higher binding affinity and cell uptake in vitro than the NGR-containing monomer, presumably due to a multivalency effect. ^99mTc-Labeled monomeric and dimeric NGR-containing peptides were subjected to SPECT imaging and biodistribution studies. SPECT scans were performed in HepG2 tumor-bearing mice at 1, 4, 12, and 24 h post-injection of ~7.4 MBq tracers. The metabolism of tracers was determined in major organs at different time points after injection which demonstrated rapid, significant tumor uptake and slow tumor washout for both traces. Predominant clearance from renal and hepatic system was also observed in ^99mTc-NGR1 and ^99mTc-NGR2. In conclusion, monomeric and dimeric NGR peptide were developed and labeled with ^99mTc successfully, while the high integrin avidity and long retention in tumor make ^99mTc-NGR dimer a promising agent for tumor angiogenesis imaging.     

Laminin promotes vascular network formation in 3D in vitro collagen scaffolds by regulating VEGF uptake

Angiogenesis is an essential neovascularisation process, which if recapitulated in 3D in vitro, will provide better understanding of endothelial cell (EC) behaviour. Various cell types and growth factors are involved, with vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 key components. We were able to control the aggregation pattern of ECs in 3D collagen hydrogels, by varying the matrix composition and/or having a source of cells signalling angiogenic proteins. These aggregation patterns reflect the different developmental pathways that ECs take to form different sized tubular structures. Cultures with added laminin and thus increased expression of α6 integrin showed a significant increase (p<0.05) in VEGFR2 positive ECs and increased VEGF uptake. This resulted in the end-to-end network aggregation of ECs. In cultures without laminin and therefore low α6 integrin expression, VEGFR2 levels and VEGF uptake were significantly lower (p<0.05). These ECs formed contiguous sheets, analogous to the ‘wrapping’ pathway in development. We have identified a key linkage between integrin expression on ECs and their uptake of VEGF, regulated by VEGFR2, resulting in different aggregation patterns in 3D.     

Preparation and biodistribution of a 99mTc tricarbonyl complex with deoxyglucose dithiocarbamate as a tumor imaging agent for SPECT

The deoxyglucose dithiocarbamate (DGDTC) was successfully labeled with the ^99mTc(CO)₃ core to provide the corresponding ^99mTc(CO)₃–DGDTC complex in good yields. The radiochemical purity of the ^99mTc(CO)₃–DGDTC complex was over 90%, as measured by high performance liquid chromatography (HPLC). The complex possessed good stability in saline at room temperature and in mouse plasma at 37 °C. Its partition coefficient result indicated that it was a hydrophilic complex. The electrophoresis results showed the complex was neutral. The biodistribution of ^99mTc(CO)₃–DGDTC in mice bearing S 180 tumor showed that the complex clearly accumulated in tumor, exhibiting high tumor/blood and tumor/muscle ratios and good tumor retention. Single photon emission computed tomography (SPECT) image studies showed there was a visible uptake in tumor sites, suggesting ^99mTc(CO)₃–DGDTC could be considered as a potential tumor imaging agent.     

Synthesis and biological evaluation of 99mTc(CO)3(His–CB) as a tumor imaging agent

The chlorambucil l-histidine conjugate was synthesized and radiolabeled with [^99mTc(CO)₃]⁺ core to form the ^99mTc(CO)₃(His–CB) complex. The radiochemical purity of the complex was over 90%. It had good hydrophilicity and was stable at room temperature. The high initial tumor uptake with certain retention, fast clearance from background, good tumor/non-tumor ratios and satisfactory scintigraphic images highlighted the potential of ^99mTc(CO)₃(His–CB) as a tumor imaging agent.     

Tetraamine‐modified octreotide and octreotate: labeling with 99mTc and preclinical comparison in AR4‐2J cells and AR4‐2J tumor‐bearing mice

Two somatostatin analogues, [^99mTc]Demotide and [^99mTc]Demotate 4, were compared with [^99mTc]Demotate 1, a previously reported somatostatin receptor subtype 2 (sst₂) targeting tracer. Conjugates were prepared by coupling an open‐chain tetraamine chelator to D ‐Phe¹ of [Tyr³]‐octreotide or [Tyr³]‐octreotate, respectively, via a p‐benzylaminodiglycolic acid spacer adopting solid‐phase peptide synthesis techniques. Peptide conjugates were collected in a highly pure form after chromatographic purification. Eventually, [^99mTc]Demotide and [^99mTc]Demotate 4 were obtained in ～1 Ci/µmol specific activity and >96% purity after labeling under alkaline conditions. Demotide and Demotate 4 exhibited similar high binding affinities for the sst₂ expressed in AR4‐2J cells with IC₅₀ values 0.16 and 0.10 nM, respectively. The (radio)metallated analogues [^99mTc]Demotide and [^99mTc]Demotate 4 showed equally high affinities to the sst₂ during saturation binding assays in AR4‐2J cell membranes (K_ds 0.08 and 0.07 nM, respectively). During incubation at 37 °C with AR4‐2J cells, the radiopeptides internalized effectively via a receptor‐mediated process, with [^99mTc]Demotate 4 exhibiting a faster internalization rate than [^99mTc]Demotide. After injection in athymic mice bearing sst₂‐expressing AR4‐2J tumors, the radiotracers showed high and specific uptake in the tumor (>25%ID/g at 1 h) and in the sst₂–positive organs. However, both [^99mTc]Demotide and [^99mTc]Demotate 4 showed unfavorably higher background activity, especially in the abdomen, in comparison to [^99mTc]Demotate 1 and are, therefore, less suited than [^99mTc]Demotate 1 for sst₂‐targeted tumor imaging in man. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.     

Selective Inhibition of Vascular Endothelial Growth Factor Receptor‐2 (VEGFR‐2) Identifies a Central Role for VEGFR‐2 in Human Aortic Endothelial Cell Responses to VEGF

Abstract

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR‐family proteins consist of VEGFR‐1/Flt‐1, VEGFR‐2/KDR/Flk‐1, and VEGFR‐3/Flt‐4. Among these, VEGFR‐2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1–3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR‐specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR‐2 tyrosine kinase, ZM323881 (5‐[[7‐(benzyloxy) quinazolin‐4‐yl]amino]‐4‐fluoro‐2‐methylphenol), to explore the role of VEGFR‐2 in endothelial cell function. Consistent with its reported effects on VEGFR‐2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR‐2, but not of VEGFR‐1, epidermal growth factor receptor (EGFR), platelet‐derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR‐1 and VEGFR‐2, but not VEGFR‐3, in the absence or presence of ZM323881. Inhibition of VEGFR‐2 blocked activation of extracellular regulated‐kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR‐1‐specific ligand, placental growth factor (PlGF). Inhibition of VEGFR‐2 also perturbed VEGF‐induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor‐2 inhibition also reversed VEGF‐stimulated phosphorylation of CrkII and its Src homology 2 (SH2)‐binding protein p130^Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR‐2 thus blocked all VEGF‐induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR‐2 is critical for VEGF signaling and/or that VEGFR‐2 may function in a heterodimer with VEGFR‐1 in human vascular endothelial cells.     

99mTc(N)-DBODC(5), a potential radiolabeled probe for SPECT of multidrug resistance: in vitro study

[^99mTc(N)(DBODC)(PNP5)]⁺ [DBODC is bis(N-ethoxyethyl)dithiocarbamato; PNP5 is bis(dimethoxypropylphosphinoethyl)ethoxyethylamine], abbreviated as ^99mTc(N)-DBODC(5), is a lipophilic cationic mixed compound investigated as a myocardial imaging agent. The findings that this tracer accumulates in mitochondrial structures through a mechanism mediated by the negative mitochondrial membrane potential and that the rapid efflux of ^99mTc(N)-DBODC(5) from nontarget tissues seems to be associated with the multidrug resistance (MDR) P-glycoprotein (P-gp) transport function open up the possibility to extend its clinical applications to tumor imaging and noninvasive MDR studies. The rate of uptake at 4 and 37 °C of ^99mTc(N)-DBODC(5) was evaluated in vitro in selected human cancer cell lines and in the corresponding sublines before and after P-gp and/or MDR-associated protein (MRP) modulator/inhibitor treatment using ^99mTc-sestamibi as a reference. The results indicated that (1) the uptake of both ^99mTc(N)-DBODC(5) and ^99mTc-sestamibi is correlated to metabolic activity of the cells and (2) the cellular accumulation is connected to the level of P-gp/MRP expression; in fact, an enhancement of uptake in resistant cells was observed after treatment with opportune MDR inhibitor/modulator, indicating that the selective blockade of P-gp/MRP prevented efflux of the tracers. This study provides a preliminary indication of the applicability of ^99mTc(N)-DBODC(5) in tumor imaging and in detecting P-gp/MRP-mediated drug resistance in human cancer. In addition, the possibility to control the hydrophobicity and pharmacological activity of this heterocomplex through the variation of the substituents on the ligands backbone without affecting the P₂S₂ coordinating sphere makes ^99mTc(N)-DBODC(5) a suitable scaffold for the preparation of a molecular probe for single photon emission computed tomography of MDR.     

VEGF receptor signalling - in control of vascular function

Vascular endothelial growth-factor receptors (VEGFRs) regulate the cardiovascular system. VEGFR1 is required for the recruitment of haematopoietic precursors and migration of monocytes and macrophages, whereas VEGFR2 and VEGFR3 are essential for the functions of vascular endothelial and lymphendothelial cells, respectively. Recent insights have shed light onto VEGFR signal transduction and the interplay between different VEGFRs and VEGF co-receptors in development, adult physiology and disease.     

99mTc标记BmK CT及其胶质瘤荷瘤裸鼠的SPECT成像研究

目的:通过放射性核素~(99m)Tc标记BmK CT多肽制备靶向胶质瘤的显像剂,探讨~(99m)?Tc-BmK CT用于胶质瘤显像的可行性。方法:采用BmK CT多肽游离的氨基与DTPA酸酐反应得到BmK CT-DTPA,经99m Tc标记后通过柱层析分离纯化制备~(99m)?Tc-BmK CT。测定标记物在PBS溶液和血清中不同时间点放射性化学纯度,评价BmK CT-~(99m)?Tc体外稳定性。新西兰白兔耳缘静脉注射~(99m)Tc-BmK CT进行SPECT显像,观察不同时间点体内的放射性分布。皮下胶质瘤裸鼠经尾静脉注射~(99m)Tc-BmK CT,观察不同时间点肿瘤的摄取情况;注射后4 h处死裸鼠,分离肿瘤和主要器官进行离体SPECT显像,并用勾画感兴趣区法分析相对放射性计数。结果:~(99m)Tc标记BmK CT多肽标记率大于80%,经柱层析分离纯化后放射性化学纯度大于99%。标记物在PBS和血清稳定性良好,6 h内放射性化学纯度均大于95%,12 h内放射性化学纯度大于90%。正常白兔SPECT显像表明~(99m)Tc-BmK CT主要浓聚在肝脏、脾脏和肾脏,软组织持续显影微弱,甲状腺区及胃肠未见核素浓聚;显像剂主要通过泌尿系统排泄,24 h肾脏与肝脏显影接近。胶质瘤裸鼠SPECT显像表明,注射后4 h肿瘤显像清楚,ROI分析结果显示肿瘤/肌肉比4.26±0.25,标记物在肿瘤内代谢缓慢,8 h肿瘤部位仍有较高摄取。结论:本研究成功制备了~(99m)Tc标记BmK CT多肽,标记物主要被肝、脾和肾摄取,经泌尿系统排泄;~(99m)Tc-BmK CT能够在皮下胶质瘤中浓聚,注射后4 h肿瘤显影清晰,瘤内代谢缓慢,有潜力成为一种新型胶质瘤分子探针。