RESUMO
PURPOSE: Overexpression of HER2/neu in breast cancer is correlated with a poor prognosis. It may vary between primary tumors and metastatic lesions and change during the treatment. Therefore, there is a need for a new means to assess HER2/neu expression in vivo. In this work, we used (68)Ga-labeled DOTA-Z(HER2:2891)-Affibody to monitor HER2/neu expression in a panel of breast cancer xenografts. METHODS: DOTA-Z(HER2:2891)-Affibody molecules were labeled with (68)Ga. In vitro binding was characterized by a receptor saturation assay. Biodistribution and PET imaging studies were conducted in athymic nude mice bearing subcutaneous human breast cancer tumors with three different levels of HER2/neu expression. Nonspecific uptake was analyzed using non-HER2-specific Affibody molecules. Signal detected by PET was compared with ex vivo assessment of the tracer uptake and HER2/neu expression. RESULTS: The (68)Ga-DOTA-Z(HER2:2891)-Affibody probe showed high binding affinity to MDA-MB-361 cells (K (D) = 1.4 ± 0.19 nM). In vivo biodistribution and PET imaging studies demonstrated high radioactivity uptake in HER2/neu-positive tumors. Tracer was eliminated quickly from the blood and normal tissues, resulting in high tumor-to-blood ratios. The highest concentration of radioactivity in normal tissue was seen in the kidneys (227 ± 14%ID/g). High-contrast PET images of HER2/neu-overexpressing tumors were recorded as soon as 1 h after tracer injection. A good correlation was observed between PET imaging, biodistribution estimates of tumor tracer concentration, and the receptor expression. CONCLUSION: These results suggest that PET imaging using (68)Ga-DOTA-Z(HER2:2891)-Affibody is sensitive enough to detect different levels of HER2/neu expression in vivo.
Assuntos
Regulação Neoplásica da Expressão Gênica , Compostos Heterocíclicos com 1 Anel/química , Tomografia por Emissão de Pósitrons/métodos , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/química , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Feminino , Radioisótopos de Gálio , Humanos , Camundongos , Imagem Multimodal , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/farmacocinética , Tomografia Computadorizada por Raios XRESUMO
The purpose of this study was to examine the melanoma imaging properties of a novel 67Ga-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (alpha-MSH) peptide. A lactam bridge-cyclized alpha-MSH peptide, DOTA-GlyGlu-CycMSH {DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]}, was synthesized and radiolabeled with 67Ga. The melanoma targeting and pharmacokinetic properties of 67Ga-DOTA-GlyGlu-CycMSH were determined in B16/F1 flank primary melanoma-bearing and B16/F10 pulmonary metastatic melanoma-bearing C57 mice. Flank primary melanoma and pulmonary metastatic melanoma imaging were performed by small animal single photon emission computed tomography (SPECT)/CT using 67Ga-DOTA-GlyGlu-CycMSH as an imaging probe. 67Ga-DOTA-GlyGlu-CycMSH was readily prepared with greater than 95% radiolabeling yield. 67Ga-DOTA-GlyGlu-CycMSH exhibited substantial tumor uptake (12.93 +/- 1.63%ID/g at 2 h postinjection) and prolonged tumor retention (5.02 +/- 1.35%ID/g at 24 h postinjection) in B16/F1 melanoma-bearing C57 mice. The uptake values for nontarget organs were generally low (<0.30%ID/g) except for the kidneys at 2, 4, and 24 h postinjection. 67Ga-DOTA-GlyGlu-CycMSH exhibited significantly (p < 0.05) higher uptakes (1.44 +/- 0.75%ID/g at 2 h postinjection and 1.49 +/- 0.69%ID/g at 4 h postinjection) in metastatic melanoma-bearing lung than those in normal lung (0.15 +/- 0.10%ID/g and 0.17 +/- 0.11%ID/g at 2 and 4 h postinjection, respectively). Both flank primary B16/F1 melanoma and B16/F10 pulmonary melanoma metastases were clearly visualized by SPECT/CT using 67Ga-DOTA-GlyGlu-CycMSH as an imaging probe 2 h postinjection. 67Ga-DOTA-GlyGlu-CycMSH exhibited favorable melanoma targeting and imaging properties, highlighting its potential as an effective imaging probe for early detection of primary and metastatic melanoma.
Assuntos
Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/secundário , Melanoma Experimental/diagnóstico por imagem , alfa-MSH , Animais , Radioisótopos de Gálio/química , Radioisótopos de Gálio/farmacocinética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Tempo , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , alfa-MSH/química , alfa-MSH/farmacocinéticaRESUMO
INTRODUCTION: Peripheral artery disease can lead to severe disability and limb loss. Therapeutic strategies focussing on macrovascular repair have shown benefit but have not significantly reduced amputation rates in progressive PAD. Proangiogenic small molecule therapies may substantially improve vascularisation in limb ischemia. The purpose of the current study was to assess the proangiogenic effects of simvastatin in a murine model of hind limb ischemia using longitudinal multimodal imaging. METHODS: Mice underwent surgical intervention to induce hind limb ischemia, and were treated with simvastatin orally for 28days. Neovascularisation was assessed using 99mTc-RGD SPECT imaging, and macrovascular volume was assessed by quantitative time of flight MRI. At each imaging time point, VEGF expression and capillary vessel density were quantified using immunohistochemical analysis. RESULTS: Simvastatin significantly increased 99mTc-RGD retention in the ischemic hind limb by day 3 post-surgery, with maximal retention at day 8. Vascular volume was significantly increased in the ischemic hind limb of simvastatin treated animals, but only by day 22. Immunohistochemical analysis shows that simvastatin significantly augmented tissue VEGF expression from day 8 with increase in capillary density (CD31+) from day 14. CONCLUSIONS: Early assessment of proangiogenic therapy efficacy can be identified using 99mTc-RGD SPECT, which displays significant increases in retention before macrovascular volume changes are measureable with MRI. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Simvastatin offers an effective proangiogenic therapy as an adjunct for management of limb ischemia. Simvastatin induces integrin expression and vascular remodeling leading to neovascularisation and improved perfusion.
Assuntos
Membro Posterior/irrigação sanguínea , Isquemia/diagnóstico por imagem , Isquemia/fisiopatologia , Imagem Multimodal , Neovascularização Fisiológica/efeitos dos fármacos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Sinvastatina/farmacologia , Animais , Capilares/efeitos dos fármacos , Capilares/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Isquemia/metabolismo , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Músculos/irrigação sanguínea , Músculos/metabolismo , Oligopeptídeos/química , Tecnécio/química , Tomografia Computadorizada de Emissão de Fóton Único , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Nanoscopy has now become a real procedure in fluorescence microscopy of living cells. The STED/RESOLFT family of nanoscopy approaches has the best prospects for delivering high speed imaging, but the history of STED includes a continuing struggle to reduce the deactivation power applied, along with difficulties in achieving simultaneous multicolor images. In this manuscript, we present a concept for a similar real-time nanoscopy, using a new class of bipartite probes that separate the luminescent and quenching functions into two coupled molecules. In particular, the STAQ (Superresolution via Transiently Activated Quencher) example we show herein employs the excited state absorbance (not ground state) of the partner to accept energy from and quench the luminescent dye. The result is that much less deactivation power is needed for superresolved (â¼50 nm) imaging. Moreover, the TAQ partner excited by the "donut" beam is shown to quench several different visible dyes via the same mechanism, opening the door to easier multicolor imaging. We demonstrate three dyes sharing the same deactivation and show examples of superresolved multicolor images. We suggest STAQ will facilitate the growth of real-time nanoscopy by reducing confounding photodamage within living cells while expanding the nanoscopist's palette.
Assuntos
Corantes Fluorescentes/análise , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Cor , LuminescênciaRESUMO
INTRODUCTION: The purpose of this study was to examine whether a novel lactam bridge-cyclized (111)In-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Gly-Glu-c[Lys-Nle-Glu-His-d-Phe-Arg-Trp-Gly-Arg-Pro-Val-Asp] {DOTA-GlyGlu-CycMSH} could be an effective imaging probe for metastatic melanoma detection. METHODS: (111)In-DOTA-GlyGlu-CycMSH was prepared and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). The internalization and efflux of (111)In-DOTA-GlyGlu-CycMSH were examined in B16/F10 melanoma cells. The biodistribution of (111)In-DOTA-GlyGlu-CycMSH was determined in B16/F10 pulmonary metastatic melanoma-bearing and normal C57 mice. Pulmonary metastatic melanoma imaging was performed by small-animal single-photon emission computed tomography (SPECT)/CT (Nano-SPECT/CT) using (111)In-DOTA-GlyGlu-CycMSH as an imaging probe and compared with 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG) positron emission tomography (PET) imaging. RESULTS: (111)In-DOTA-GlyGlu-CycMSH was readily prepared with greater than 95% radiolabeling yield. (111)In-DOTA-GlyGlu-CycMSH displayed rapid internalization and extended efflux in B16/F10 cells. (111)In-DOTA-GlyGlu-CycMSH exhibited significantly (P<.05) higher uptakes (2.00+/-0.74%ID/g at 2 h post-injection and 1.83+/-0.12%ID/g at 4 h post-injection) in metastatic melanoma-bearing lung than that in normal lung (0.08+/-0.08%ID/g and 0.05+/-0.05%ID/g at 2 and 4 h post-injection, respectively). The activity accumulation in normal organs was low (<0.5%ID/g) except for the kidneys 2 and 4 h post-injection. B16/F10 pulmonary melanoma metastases were clearly visualized with (111)In-DOTA-GlyGlu-CycMSH 2 h post-injection rather than with [(18)F]FDG 1 h post-injection. CONCLUSIONS: (111)In-DOTA-GlyGlu-CycMSH exhibited favorable metastatic melanoma-targeting and -imaging properties, highlighting its potential as an effective imaging probe for metastatic melanoma detection.
Assuntos
Lactamas/química , Melanoma/diagnóstico , Melanoma/patologia , Metástase Neoplásica/diagnóstico , Peptídeos Cíclicos , alfa-MSH , Sequência de Aminoácidos , Animais , Ligação Competitiva , Linhagem Celular Tumoral , Estudos de Viabilidade , Fluordesoxiglucose F18/administração & dosagem , Compostos Heterocíclicos com 1 Anel/química , Radioisótopos de Índio , Melanoma/diagnóstico por imagem , Camundongos , Metástase Neoplásica/diagnóstico por imagem , Estadiamento de Neoplasias , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacocinética , Reprodutibilidade dos Testes , Coloração e Rotulagem , Fatores de Tempo , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , alfa-MSH/química , alfa-MSH/metabolismo , alfa-MSH/farmacocinéticaRESUMO
A new antisense peptide-peptide nucleic acid (peptide-PNA) conjugate, designed for targeting bcl-2 expression, has been radiolabeled, characterized, and evaluated for bcl-2 mRNA binding in a cell-free system. A PNA complementary to the first six codons of the bcl-2 gene was synthesized by standard solid-phase Fmoc chemistry and conjugated to a new derivative of 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA) that allows macrocyclic radiometal chelates to be incorporated into any sequence position of a peptide-PNA conjugate. The DOTA-PNA conjugate was then coupled to a membrane-permeating transduction peptide, PTD-4, designed for intracellular delivery of the radiolabeled PNA. The conjugate was characterized by HPLC and ESI-MS and labeled with (111)In and (90)Y to high specific activities (>1000 Ci/mmol) with high radiochemical purity. Northern blot analysis showed that (90)Y-PTD-4-K(DOTA)-anti-bcl-2-PNA bound specifically to as little as 50 fmol of bcl-2 mRNA, a result equivalent to that obtained with the analogous (32)P-labeled DNA antisense oligonucleotide. Thus, the mRNA targeting properties of (111)In- and (90)Y-PTD-4-K(DOTA)-anti-bcl-2-PNA demonstrate potential for diagnostic imaging and targeted radiotherapy applications in bcl-2-positive cancers.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes bcl-2/genética , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/farmacologia , Peptídeos/síntese química , Peptídeos/farmacologia , RNA Mensageiro/metabolismo , Sequência de Bases , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Radioisótopos de Índio , Ácidos Nucleicos Peptídicos/genética , Ácidos Nucleicos Peptídicos/metabolismo , Peptídeos/metabolismo , RNA Mensageiro/genética , Radioisótopos de ÍtrioRESUMO
The B-cell lymphoma/leukemia-2 (bcl-2) proto-oncogene has been associated with the transformation of benign lesions to malignancy, disease progression, poor prognosis, reduced survival, and development of resistance to radiation and chemotherapy in many types of cancer. The objective of this work was to synthesize an antisense peptide nucleic acid (PNA) complementary to the first six codons of the bcl-2 open reading frame, conjugated to a membrane-permeating peptide for intracellular delivery, and modified with a bifunctional chelating agent for targeting imaging and therapeutic radiometals to tumors overexpressing bcl-2. Four peptide-PNA constructs were synthesized by a combination of manual and automated stepwise elongation techniques, including bcl-2 antisense conjugates and nonsense conjugates with no complementarity to any known mammalian gene or DNA sequence. The PNA sequences were synthesized manually by solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) techniques. Then a fully protected lysine monomer, modified with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'"-tetraacetic acid (DOTA) for radiometal chelation, was coupled manually to each PNA sequence. Synthesis of the DOTA-PNA conjugates was followed by automated elongation with a peptide sequence (PTD-4-glycine, PTD-4-G), known to mediate cellular internalization of impermeable effector molecules, or its retro-inverso analogue (ri-PTD-4-G). Preparation of the four conjugates required an innovative synthetic strategy, using mild acid conditions to generate hydrophobic, partially deprotected intermediates. These intermediates were purified by semipreparative reversed-phase HPLC and completely deprotected to yield pure peptide-PNA conjugates in 6% to 9% overall yield. Using modifications of this synthetic strategy, the ri-PTD-4-G conjugate of bcl-2 antisense PNA was prepared using a lysine derivative of tetramethylrhodamine (TMR) for fluorescence microscopy. Plasma stability studies showed that (111)In-DOTA-labeled ri-PTD-4-G-anti-bcl-2 PNA was stable for 168 h at 37 degrees C, unlike the conjugate containing the parent peptide sequence. Scanning confocal fluorescence microscopy of TMR-labeled ri-PTD-4-G-anti-bcl-2 PNA in Raji lymphoma cells demonstrated that the retro-inverso peptide was active in membrane permeation and mediated cellular internalization of the antisense PNA into the cytoplasm, where high concentrations of bcl-2 mRNA are expected to be present.