RESUMO
BACKGROUND: Tumor-specific biomarkers are a prerequisite for the development of targeted imaging and therapy in oral squamous cell carcinoma (OSCC). urokinase-type Plasminogen Activator Receptor (uPAR), Tissue Factor (TF) and Epidermal Growth Factor Receptor (EGFR) are three biomarkers that exhibit enhanced expression in many types of cancers, and have been investigated as potential biomarkers for targeted strategies and prognostication. The aim of the study was to investigate the expression patterns of uPAR, TF and EGFR and their potential prognostic value in OSCC. METHODS: Immunohistochemical expression of uPAR, TF and EGFR in tumor resection specimens from 191 patients with primary OSCC was analyzed. Overall (OS) and disease-free survival (DFS) was calculated. Associations between biomarker expression, clinicopathological factors and patient survival was analyzed using the Cox proportional hazards model for univariate and multivariate analysis, log rank and Kaplan-Meier statistics. RESULTS: uPAR and TF exhibited a highly tumor-specific expression pattern while EGFR also showed expression in normal tissues outside the tumor compartment. The overall positive expression rate of uPAR, TF and EGFR was 95%, 58% and 98%, respectively. High uPAR expression across the entire cohort was negatively associated with OS (p = 0.031, HR = 1.595 (95%CI 1.044-2.439)) in univariate analysis. The 5-year OS for high and low uPAR expression was 39% and 56%, respectively. The expression of TF and EGFR was not associated with survival outcome. CONCLUSIONS: This study may suggest that uPAR and TF could potentially be attractive targets for molecular imaging and therapy in OSCC due to high positive expression rates and tumor-specific expression patterns. High uPAR expression was significantly associated with a reduced survival. uPAR seems to be a prognostic biomarker in oral cancer.
Assuntos
Apoenzimas/genética , Carcinoma de Células Escamosas/terapia , Receptores ErbB/genética , Neoplasias Bucais/terapia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Tromboplastina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoenzimas/análise , Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/metabolismo , Intervalo Livre de Doença , Receptores ErbB/análise , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Modelos de Riscos Proporcionais , Receptores de Ativador de Plasminogênio Tipo Uroquinase/análise , Tromboplastina/análise , Adulto JovemRESUMO
In osteosarcoma, a primary mesenchymal bone cancer occurring predominantly in younger patients, invasive tumour growth leads to extensive bone destruction. This process is insufficiently understood, cannot be efficiently counteracted and calls for novel means of treatment. The endocytic collagen receptor, uPARAP/Endo180, is expressed on various mesenchymal cell types and is involved in bone matrix turnover during normal bone growth. Human osteosarcoma specimens showed strong expression of this receptor on tumour cells, along with the collagenolytic metalloprotease, MT1-MMP. In advanced tumours with ongoing bone degeneration, sarcoma cells positive for these proteins formed a contiguous layer aligned with the degradation zones. Remarkably, osteoclasts were scarce or absent from these regions and quantitative analysis revealed that this scarcity marked a strong contrast between osteosarcoma and bone metastases of carcinoma origin. This opened the possibility that sarcoma cells might directly mediate bone degeneration. To examine this question, we utilized a syngeneic, osteolytic bone tumour model with transplanted NCTC-2472 sarcoma cells in mice. When analysed in vitro, these cells were capable of degrading the protein component of surface-labelled bone slices in a process dependent on MMP activity and uPARAP/Endo180. Systemic treatment of the sarcoma-inoculated mice with a mouse monoclonal antibody that blocks murine uPARAP/Endo180 led to a strong reduction of bone destruction. Our findings identify sarcoma cell-resident uPARAP/Endo180 as a central player in the bone degeneration of advanced tumours, possibly following an osteoclast-mediated attack on bone in the early tumour stage. This points to uPARAP/Endo180 as a promising therapeutic target in osteosarcoma, with particular prospects for improved neoadjuvant therapy.
Assuntos
Neoplasias Ósseas/patologia , Osteólise/metabolismo , Osteossarcoma/patologia , Receptores Mitogênicos/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Invasividade Neoplásica , Osteoclastos/patologia , Osteólise/etiologia , Osteólise/patologiaRESUMO
The urokinase-type plasminogen activator receptor (uPAR) is implicated in cancer invasion and metastatic development in prostate cancer and provides therefore an attractive molecular target for both imaging and therapy. In this study, we provide the first in vivo data on an antimetastatic effect of uPAR radionuclide targeted therapy in such lesions and show the potential of uPAR positron emission tomography (PET) imaging for identifying small foci of metastatic cells in a mouse model of disseminating human prostate cancer. Two radiolabeled ligands were generated in high purity and specific activity: a uPAR-targeting probe ((177)Lu-DOTA-AE105) and a nonbinding control ((177)Lu-DOTA-AE105mut). Both uPAR flow cytometry and ELISA confirmed high expression levels of the target uPAR in PC-3M-LUC2.luc cells, and cell binding studies using (177)Lu-DOTA-AE105 resulted in a specific binding with an IC50 value of 100 nM in a competitive binding experiment. In vivo, uPAR targeted radionuclide therapy significantly reduced the number of metastatic lesions in the disseminated metastatic prostate cancer model, when compared to vehicle and nontargeted (177)Lu groups (p < 0.05) using bioluminescence imaging. Moreover, we found a significantly longer metastatic-free survival, with 65% of all mice without any disseminated metastatic lesions present at 65 days after first treatment dose (p = 0.047). In contrast, only 30% of all mice in the combined control groups treated with (177)Lu-DOTA-AE105mut or vehicle were without metastatic lesions. No treatment-induced toxicity was observed during the study as evaluated by observing animal weight and H&E staining of kidney tissue (dose-limiting organ). Finally, uPAR PET imaging using (64)Cu-DOTA-AE105 detected all small, disseminated metastatic foci when compared with bioluminescence imaging in a cohort of animals during the treatment study. In conclusion, uPAR targeted radiotherapy resulted in a significant reduction in the number of metastatic lesions in a human metastatic prostate cancer model. Furthermore, we have provided the first evidence of the potential for identification of small metastatic lesions using uPAR PET imaging in disseminated prostate cancer, illustrating the promising strategy of uPAR theranostics in prostate cancer.
Assuntos
Complexos de Coordenação/química , Lutécio/química , Oligopeptídeos/química , Neoplasias da Próstata/radioterapia , Radioisótopos/uso terapêutico , Animais , Linhagem Celular Tumoral , Intervalo Livre de Doença , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Ligantes , Masculino , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Tomografia por Emissão de Pósitrons , Neoplasias da Próstata/diagnóstico por imagem , Ligação Proteica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Proteínas Recombinantes/química , Resultado do Tratamento , Microtomografia por Raio-XRESUMO
Histone deacetylase (HDAC) inhibition is a novel entity in medical oncology, and several HDAC inhibitors are in clinical trials. One of them is the hydroxamic acid belinostat (PXD101) that has demonstrated therapeutic efficacy for several clinical indications. Acetylation of histones is a key event after treatment with HDAC inhibitors, and could thus be used as a marker for monitoring cellular response to HDAC inhibitor treatment. Here we describe the utility of a newly described monoclonal antibody against acetylated H4 for immunohistochemistry on paraffin-embedded fine needle biopsies from nude mice carrying A2780 human ovarian cancer xenografts. Acetylated H4 was monitored in vivo by immunohistochemistry during treatment with belinostat, and compared with pharmacokinetics in plasma and tumor tissue. We found an increased level of acetylated H4 15 min after a single treatment (200 mg/kg i.v.) with maximum level reached after 1 h. H4 acetylation intensity reflected the belinostat concentration in plasma and tumor tissue. The threshold level for belinostat activity, indicated by acetylated H4, correlated with belinostat plasma concentrations above 1,000 ng/ml. In conclusion, examination of H4 acetylation in fine needle biopsies using the T25 antibody may prove useful in monitoring HDAC inhibitor efficacy in clinical trials involving humans with solid tumors.
Assuntos
Antineoplásicos/uso terapêutico , Histonas/metabolismo , Ácidos Hidroxâmicos/uso terapêutico , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Acetilação/efeitos dos fármacos , Animais , Anticorpos Monoclonais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Feminino , Células HCT116 , Inibidores de Histona Desacetilases , Histona Desacetilases/imunologia , Histonas/imunologia , Humanos , Ácidos Hidroxâmicos/sangue , Ácidos Hidroxâmicos/farmacocinética , Leucemia P388/tratamento farmacológico , Leucemia P388/enzimologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/enzimologia , Masculino , Camundongos , Camundongos Nus , Neoplasias Experimentais/enzimologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Sulfonamidas , Transplante HeterólogoRESUMO
Urokinase-type plasminogen activator receptor (uPAR) overexpression is an important biomarker for aggressiveness in cancer including prostate cancer (PC) and provides independent clinical information in addition to prostate-specific antigen and Gleason score. This article focuses on uPAR PET as a new diagnostic and prognostic imaging biomarker in PC. Many preclinical uPAR-targeted PET imaging studies using AE105 in cancer models have been undertaken with promising results. A major breakthrough was obtained with the recent human translation of uPAR PET in using 64Cu- and 68Ga-labelled versions of AE105, respectively. Clinical results from patients with PC included in these studies are encouraging and support continuation with large-scale clinical trials.
Assuntos
Neoplasias da Próstata/diagnóstico por imagem , Compostos Radiofarmacêuticos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Radioisótopos de Gálio , Compostos Heterocíclicos , Compostos Heterocíclicos com 1 Anel , Humanos , Masculino , Camundongos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada por Raios XRESUMO
Urokinase plasminogen activator receptor (uPAR) is a key component in proteolysis and extracellular matrix degradation during cancer invasion and metastasis. uPAR overexpression is an important biomarker for aggressiveness in several solid tumors and provides independent clinical information. A recent major breakthrough was obtained with human translation of uPAR PET using 68Ga-NOTA-AE105. Clinical results are encouraging and several large-scale clinical trials are now ongoing. This review focuses on uPAR PET with 68Ga-NOTA-AE105 as a new broadly applicable diagnostic and prognostic imaging biomarker in cancer.
Assuntos
Radioisótopos de Gálio , Ligantes , Neoplasias/química , Neoplasias/diagnóstico por imagem , Receptores de Ativador de Plasminogênio Tipo Uroquinase/análise , Humanos , Tomografia por Emissão de Pósitrons , Cintilografia/métodos , Compostos Radiofarmacêuticos , Tomografia Computadorizada por Raios XRESUMO
PURPOSE: Urokinase-like Plasminogen Activator Receptor (uPAR) is overexpressed in a variety of carcinoma types, and therefore represents an attractive imaging target. The aim of this study was to assess the feasibility of two uPAR-targeted probes for PET and fluorescence tumor imaging in a human xenograft tongue cancer model. EXPERIMENTAL DESIGN AND RESULTS: Tumor growth of tongue cancer was monitored by bioluminescence imaging (BLI) and MRI. Either ICG-Glu-Glu-AE105 (fluorescent agent) or 64Cu-DOTA-AE105 (PET agent) was injected systemically, and fluorescence imaging or PET/CT imaging was performed. Tissue was collected for micro-fluorescence imaging and histology. A clear fluorescent signal was detected in the primary tumor with a mean in vivo tumor-to-background ratio of 2.5. Real-time fluorescence-guided tumor resection was possible, and sub-millimeter tumor deposits could be localized. Histological analysis showed co-localization of the fluorescent signal, uPAR expression and tumor deposits. In addition, the feasibility of uPAR-guided robotic cancer surgery was demonstrated. Also, uPAR-PET imaging showed a clear and localized signal in the tongue tumors. CONCLUSIONS: This study demonstrated the feasibility of combining two uPAR-targeted probes in a preclinical head and neck cancer model. The PET modality provided preoperative non-invasive tumor imaging and the optical modality allowed for real-time fluorescence-guided tumor detection and resection. Clinical translation of this platform seems promising.
Assuntos
Neoplasias de Cabeça e Pescoço/cirurgia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/análise , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Cirurgia Assistida por Computador/métodos , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/cirurgia , Linhagem Celular Tumoral , Estudos de Viabilidade , Feminino , Corantes Fluorescentes/química , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos Endogâmicos , Camundongos Nus , Imagem Óptica/métodos , Reprodutibilidade dos Testes , Neoplasias da Língua/diagnóstico por imagem , Neoplasias da Língua/metabolismo , Neoplasias da Língua/cirurgiaRESUMO
The overexpression of urokinase-type plasminogen activator receptors (uPARs) represents an established biomarker for aggressiveness in most common malignant diseases, including breast cancer (BC), prostate cancer (PC), and urinary bladder cancer (UBC), and is therefore an important target for new cancer therapeutic and diagnostic strategies. In this study, uPAR PET imaging using a 68Ga-labeled version of the uPAR-targeting peptide (AE105) was investigated in a group of patients with BC, PC, and UBC. The aim of this first-in-human, phase I clinical trial was to investigate the safety and biodistribution in normal tissues and uptake in tumor lesions. Methods: Ten patients (6 PC, 2 BC, and 2 UBC) received a single intravenous dose of 68Ga-NOTA-AE105 (154 ± 59 MBq; range, 48-208 MBq). The biodistribution and radiation dosimetry were assessed by serial whole-body PET/CT scans (10 min, 1 h, and 2 h after injection). Safety assessment included measurements of vital signs with regular intervals during the imaging sessions and laboratory blood screening tests performed before and after injection. In a subgroup of patients, the in vivo stability of 68Ga-NOTA-AE105 was determined in collected blood and urine. PET images were visually analyzed for visible tumor uptake of 68Ga-NOTA-AE105, and SUVs were obtained from tumor lesions by manually drawing volumes of interest in the malignant tissue. Results: No adverse events or clinically detectable pharmacologic effects were found. The radioligand exhibited good in vivo stability and fast clearance from tissue compartments primarily by renal excretion. The effective dose was 0.015 mSv/MBq, leading to a radiation burden of 3 mSv when the clinical target dose of 200 MBq was used. In addition, radioligand accumulation was seen in primary tumor lesions as well as in metastases. Conclusion: This first-in-human, phase I clinical trial demonstrates the safe use and clinical potential of 68Ga-NOTA-AE105 as a new radioligand for uPAR PET imaging in cancer patients.
Assuntos
Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Oligopeptídeos/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Exposição à Radiação/análise , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Feminino , Compostos Heterocíclicos/farmacocinética , Compostos Heterocíclicos com 1 Anel , Humanos , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Imagem Molecular/métodos , Especificidade de Órgãos , Projetos Piloto , Doses de Radiação , Exposição à Radiação/prevenção & controle , Compostos Radiofarmacêuticos/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Distribuição Tecidual , Contagem Corporal TotalRESUMO
Overexpression of urokinase-type plasminogen activator receptors (uPAR) represents an important biomarker for aggressiveness in most common malignant diseases, including prostate cancer (PC). Accordingly, uPAR expression either assessed directly in malignant PC tissue or assessed directly in plasma (intact/cleaved forms)-provides independent additional clinical information to that contributed by PSA, Gleason score, and other relevant pathological and clinical parameters. In this respect, non-invasive molecular imaging by positron emission tomography (PET) offers a very attractive technology platform, which can provide the required quantitative information on the uPAR expression profile, without the need for invasive procedures and the risk of missing the target due to tumor heterogeneity. These observations support non-invasive PET imaging of uPAR in PC as a clinically relevant diagnostic and prognostic imaging method. In this review, we will focus on the recent development of uPAR PET and the relevance within prostate cancer imaging. Novel antibody and small-molecule radiotracers-targeting uPAR, including a series of uPAR-targeting PET ligands, based on the high affinity peptide ligand AE105, have been synthesized and tested in vitro and in vivo in preclinical murine xenograft models and, recently, in a first-ever clinical uPAR PET study in cancer patients, including patients with PC. In this phase I study, a high and specific uptake of the tracer 64Cu-DOTA-AE105 was found in both primary tumors and lymph node metastases. The results are encouraging and support large-scale clinical trials to determine the utility of uPAR PET in the management of patients with PC with the goal of improving outcome.
RESUMO
Near infrared intra-operative optical imaging is an emerging technique with clear implications for improved cancer surgery by enabling a more distinct delineation of the tumor margins during resection. This modality has the potential to increase the number of patients having a curative radical tumor resection. In the present study, a new uPAR-targeted fluorescent probe was developed and the in vivo applicability was evaluated in a human xenograft mouse model. Most human carcinomas express high level of uPAR in the tumor-stromal interface of invasive lesions and uPAR is therefore considered an ideal target for intra-operative imaging. Conjugation of the flourophor indocyanine green (ICG) to the uPAR agonist (AE105) provides an optical imaging ligand with sufficiently high receptor affinity to allow for a specific receptor targeting in vivo. For in vivo testing, human glioblastoma xenograft mice were subjected to optical imaging after i.v. injection of ICG-AE105, which provided an optimal contrast in the time window 6-24 h post injection. Specificity of the uPAR-targeting probe ICG-AE105 was demonstrated in vivo by 1) no uptake of unconjugated ICG after 15 hours, 2) inhibition of ICG-AE105 tumor uptake by a bolus injection of the natural uPAR ligand pro-uPA, and finally 3) the histological colocalization of ICG-AE105 fluorescence and immunohistochemical detected human uPAR on resected tumor slides. Taken together, our data supports the potential use of this probe for intra-operative optical guidance in cancer surgery to ensure complete removal of tumors while preserving adjacent, healthy tissue.
Assuntos
Diagnóstico por Imagem , Dipeptídeos/química , Verde de Indocianina/química , Neoplasias/cirurgia , Oligopeptídeos/química , Peptídeos/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Corantes Fluorescentes , Humanos , Ligantes , Camundongos , Camundongos Nus , Imagem Óptica , Estabilidade ProteicaRESUMO
UNLABELLED: Glioblastoma is one of the most malignant types of human cancer, and the prognosis is poor. The development and validation of novel molecular imaging biomarkers has the potential to improve tumor detection, grading, risk stratification, and treatment monitoring of gliomas. The aim of this study was to explore the potential of PET imaging of the urokinase-type plasminogen activator receptor (uPAR) in glioblastoma. METHODS: The uPAR messenger RNA expression of tumors from 19 glioblastoma patients was analyzed, and a cell culture derived from one of these patients was used to establish an orthotopic xenograft model of glioblastoma. Tumor growth was monitored using bioluminescence imaging. Five to six weeks after inoculation, all mice were scanned with small-animal PET/CT using two new uPAR PET ligands ((64)Cu-NOTA-AE105 and (68)Ga-NOTA-AE105) and, for comparison, O-(2-(18)F-fluoroethyl)-l-tyrosine ((18)F-FET). One MRI scan was obtained for each mouse to confirm tumor location. The uPAR specificity of (64)Cu-NOTA-AE105 was confirmed by alignment of hematoxylin- and eosin-stained and uPAR immunohistochemistry-stained slides of the brain with the activity distribution as determined using autoradiography. RESULTS: uPAR expression was found in all 19 glioblastoma patient tumors, and high expression of uPAR correlated with decreased overall survival (P = 0.04). Radiolabeling of NOTA-AE105 with (64)Cu and (68)Ga was straightforward, resulting in a specific activity of approximately 20 GBq/µmol and a radiochemical purity of more than 98% for (64)Cu-NOTA-AE105 and more than 97% for (68)Ga-NOTA-AE105. High image contrast resulting in clear tumor delineation was found for both (68)Ga-NOTA-AE105 and (64)Cu-NOTA-AE105. Absolute uptake in tumor was higher for (18)F-FET (3.5 ± 0.8 percentage injected dose [%ID]/g) than for (64)Cu-NOTA-AE105 (1.2 ± 0.4 %ID/g) or (68)Ga-NOTA-AE105 (0.4 ± 0.1 %ID/g). A similar pattern was observed in background brain tissue, where uptake was 1.9 ± 0.1 %ID/g for (18)F-fluorothymidine, compared with 0.05 ± 0.01 %ID/g for (68)Ga-NOTA-AE105 and 0.11 ± 0.02 %ID/g for (64)Cu-NOTA-AE105. The result was a significantly higher tumor-to-background ratio for both (68)Ga-NOTA-AE105 (7.6 ± 2.1, P < 0.05) and (64)Cu-NOTA-AE105 (10.6 ± 2.3, P < 0.01) than for (18)F-FET PET (1.8 ± 0.3). Autoradiography of brain slides confirmed that the accumulation of (64)Cu-NOTA-AE105 corresponded well with uPAR-positive cancer cells. CONCLUSION: On the basis of our translational study, uPAR PET may be a highly promising imaging biomarker for glioblastoma. Further clinical exploration of uPAR PET in glioblastoma is therefore justified.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Glioblastoma/diagnóstico por imagem , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adulto , Animais , Autorradiografia , Biomarcadores Tumorais , Células Cultivadas , Radioisótopos de Cobre , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons , RNA Mensageiro/biossíntese , Compostos Radiofarmacêuticos , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
C4.4A is a modular glycolipid-anchored Ly6/uPAR/α-neurotoxin multidomain protein that exhibits a prominent membrane-associated expression in stratified squamous epithelia. C4.4A is also expressed in various solid cancer lesions, where high expression levels often are correlated to poor prognosis. Circumstantial evidence suggests a role for C4.4A in cell adhesion, migration, and invasion, but a well-defined biological function is currently unknown. In the present study, we have generated and characterized the first C4.4A-deficient mouse line to gain insight into the functional significance of C4.4A in normal physiology and cancer progression. The unchallenged C4.4A-deficient mice were viable, fertile, born in a normal Mendelian distribution and, surprisingly, displayed normal development of squamous epithelia. The C4.4A-deficient mice were, nonetheless, significantly lighter than littermate controls predominantly due to differences in fat mass. Congenital C4.4A deficiency delayed migration of keratinocytes enclosing incisional skin wounds in male mice. In chemically induced bladder carcinomas, C4.4A deficiency attenuated the incidence of invasive lesions despite having no effect on total tumour burden. This new C4.4A-deficient mouse line provides a useful platform for future studies on functional aspects of C4.4A in tumour cell invasion in vivo.
Assuntos
Moléculas de Adesão Celular/genética , Epiderme/embriologia , Epiderme/metabolismo , Proteínas Ligadas por GPI/genética , Deleção de Genes , Animais , Peso Corporal , Carcinoma Pulmonar de Lewis , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/metabolismo , Metabolismo Energético/genética , Epiderme/ultraestrutura , Epitélio/ultraestrutura , Feminino , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Imageamento por Ressonância Magnética , Masculino , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fenótipo , Frações Subcelulares/metabolismo , Magreza/metabolismo , Tomografia Computadorizada por Raios X , Bexiga Urinária/patologia , Perda Insensível de Água , CicatrizaçãoRESUMO
A first-in-human clinical trial with Positron Emission Tomography (PET) imaging of the urokinase-type plasminogen activator receptor (uPAR) in patients with breast, prostate and bladder cancer, is described. uPAR is expressed in many types of human cancers and the expression is predictive of invasion, metastasis and indicates poor prognosis. uPAR PET imaging therefore holds promise to be a new and innovative method for improved cancer diagnosis, staging and individual risk stratification. The uPAR specific peptide AE105 was conjugated to the macrocyclic chelator DOTA and labeled with (64)Cu for targeted molecular imaging with PET. The safety, pharmacokinetic, biodistribution profile and radiation dosimetry after a single intravenous dose of (64)Cu-DOTA-AE105 were assessed by serial PET and computed tomography (CT) in 4 prostate, 3 breast and 3 bladder cancer patients. Safety assessment with laboratory blood screening tests was performed before and after PET ligand injection. In a subgroup of the patients, the in vivo stability of our targeted PET ligand was determined in collected blood and urine. No adverse or clinically detectable side effects in any of the 10 patients were found. The ligand exhibited good in vivo stability and fast clearance from plasma and tissue compartments by renal excretion. In addition, high uptake in both primary tumor lesions and lymph node metastases was seen and paralleled high uPAR expression in excised tumor tissue. Overall, this first-in-human study therefore provides promising evidence for safe use of (64)Cu-DOTA-AE105 for uPAR PET imaging in cancer patients.
Assuntos
Neoplasias da Mama/diagnóstico , Imagem Molecular/métodos , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Próstata/diagnóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/análise , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Mama/patologia , Radioisótopos de Cobre/administração & dosagem , Radioisótopos de Cobre/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Feminino , Compostos Heterocíclicos com 1 Anel/administração & dosagem , Compostos Heterocíclicos com 1 Anel/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Oligopeptídeos/administração & dosagem , Oligopeptídeos/metabolismo , Prognóstico , Neoplasias da Próstata/patologia , Ligação Proteica , Neoplasias da Bexiga Urinária/patologiaRESUMO
UNLABELLED: (64)Cu-DOTA-AE105 is a novel positron emission tomography (PET) tracer specific to the human urokinase-type plasminogen activator receptor (uPAR). In preparation of using this tracer in humans, as a new promising method to distinguish between indolent and aggressive cancers, we have performed PET studies in mice to evaluate the in vivo biodistribution and estimate human dosimetry of (64)Cu-DOTA-AE105. METHODS: Five mice received iv tail injection of (64)Cu-DOTA-AE105 and were PET/CT scanned 1, 4.5 and 22 h post injection. Volume-of-interest (VOI) were manually drawn on the following organs: heart, lung, liver, kidney, spleen, intestine, muscle, bone and bladder. The activity concentrations in the mentioned organs [%ID/g] were used for the dosimetry calculation. The %ID/g of each organ at 1, 4.5 and 22 h was scaled to human value based on a difference between organ and body weights. The scaled values were then exported to OLINDA software for computation of the human absorbed doses. The residence times as well as effective dose equivalent for male and female could be obtained for each organ. To validate this approach, of human projection using mouse data, five mice received iv tail injection of another (64)Cu-DOTA peptide-based tracer, (64)Cu-DOTA-TATE, and underwent same procedure as just described. The human dosimetry estimates were then compared with observed human dosimetry estimate recently found in a first-in-man study using (64)Cu-DOTA-TATE. RESULTS: Human estimates of (64)Cu-DOTA-AE105 revealed the heart wall to receive the highest dose (0.0918 mSv/MBq) followed by the liver (0.0815 mSv/MBq), All other organs/tissue were estimated to receive doses in the range of 0.02-0.04 mSv/MBq. The mean effective whole-body dose of (64)Cu-DOTA-AE105 was estimated to be 0.0317 mSv/MBq. Relatively good correlation between human predicted and observed dosimetry estimates for (64)Cu-DOTA-TATE was found. Importantly, the effective whole body dose was predicted with very high precision (predicted value: 0.0252 mSv/Mbq, Observed value: 0.0315 mSv/MBq) thus validating our approach for human dosimetry estimation. CONCLUSION: Favorable dosimetry estimates together with previously reported uPAR PET data fully support human testing of (64)Cu-DOTA-AE105.
Assuntos
Radioisótopos de Cobre , Peptídeos/química , Tomografia por Emissão de Pósitrons/métodos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Feminino , Humanos , Masculino , Camundongos , Peptídeos/farmacocinética , Traçadores Radioativos , RadiometriaRESUMO
Urokinase-type plasminogen activator receptor (uPAR) has been shown to be of special importance during cancer invasion and metastasis. However, currently, tissue samples are needed for measurement of uPAR expression limiting the potential as a clinical routine. Therefore, non-invasive methods are needed. In line with this, uPAR has recently been identified as a very promising imaging target candidate. uPAR consists of three domains attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor and binds it natural ligand uPA with high affinity to localize plasminogen activation at the cell surface. Due to the importance of uPAR in cancer invasion and metastasis, a number of high-affinity ligands have been identified during the last decades. These ligands have recently been used as starting point for the development of a number of ligands for imaging of uPAR using various imaging modalities such as optical imaging, magnetic resonance imaging, single photon emission computer tomography (SPECT) and positron emission topography (PET). In this review, we will discuss recent advances in the development of uPAR-targeted imaging ligands according to imaging modality. In addition, we will discuss the potential future clinical application for uPAR imaging as a new imaging biomarker.
Assuntos
Biomarcadores Tumorais/análise , Imagem Molecular , Sondas Moleculares , Neoplasias/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/análise , Animais , Humanos , Ligantes , Imagem Molecular/métodos , Neoplasias/patologia , Valor Preditivo dos Testes , PrognósticoRESUMO
UNLABELLED: Urokinase-type plasminogen activator receptor (uPAR) is overexpressed in human prostate cancer and uPAR has been found to be associated with metastatic disease and poor prognosis. AE105 is a small linear peptide with high binding affinity to uPAR. We synthesized an N-terminal NOTA-conjugated version (NOTA-AE105) for development of the first (18)F-labeled uPAR positron-emission-tomography PET ligand using the Al(18)F radiolabeling method. In this study, the potential of (18)F-AlF-NOTA-AE105 to specifically target uPAR-positive prostate tumors was investigated. METHODS: NOTA-conjugated AE105 was synthesized and radiolabeled with (18)F-AlF according to a recently published optimized protocol. The labeled product was purified by reverse phase high performance liquid chromatography RP-HPLC. The tumor targeting properties were evaluated in mice with subcutaneously inoculated PC-3 xenografts using small animal PET and ex vivo biodistribution studies. uPAR-binding specificity was studied by coinjection of an excess of a uPAR antagonist peptide AE105 analogue (AE152). RESULTS: NOTA-AE105 was labeled with (18)F-AlF in high radiochemical purity (>92%) and yield (92.7%) and resulted in a specific activity of greater than 20GBq/µmol. A high and specific tumor uptake was found. At 1h post injection, the uptake of (18)F-AlF-NOTA-AE105 in PC-3 tumors was 4.22 ± 0.13%ID/g. uPAR-binding specificity was demonstrated by a reduced uptake of (18)F-AlF-NOTA-AE105 after coinjection of a blocking dose of uPAR antagonist at all three time points investigated. Good tumor-to-background ratio was observed with small animal PET and confirmed in the biodistribution analysis. Ex vivo uPAR expression analysis on extracted tumors confirmed human uPAR expression that correlated close with tumor uptake of (18)F-AlF-NOTA-AE105. CONCLUSION: The first (18)F-labeled uPAR PET ligand, (18)F-AlF-NOTA-AE105, has successfully been prepared and effectively visualized noninvasively uPAR positive prostate cancer. The favorable in vivo kinetics and easy production method facilitate its future clinical translation for identification of prostate cancer patients with an invasive phenotype and poor prognosis.
Assuntos
Transformação Celular Neoplásica , Radioisótopos de Flúor , Oligopeptídeos , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Próstata/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Compostos Heterocíclicos/química , Compostos Heterocíclicos com 1 Anel , Humanos , Marcação por Isótopo , Ligantes , Masculino , Camundongos , Oligopeptídeos/química , Oligopeptídeos/farmacocinética , Neoplasias da Próstata/diagnóstico por imagem , RadioquímicaRESUMO
INTRODUCTION: The use of copper-based positron emission tomography (PET) tracers in cancer studies is increasing. However, as copper has previously been found in high concentrations in human tumor tissue in vivo, instability of PET tracers could result in tumor accumulation of non-tracer-bound radioactive copper that may influence PET measurements. Here we determine the degree of (64)Cu uptake in five commonly used human cancer xenograft models in mice. Additionally, we compare copper accumulation in tumor tissue to gene expression of human copper transporter 1 (CTR1). METHODS: Small animal PET scans were performed on five different human cancer xenograft mice models 1h and 22h post injection (p.i.) of (64)CuCl2. Regions of interest (ROIs) were drawn on tumor tissue and sections of various organs on all images. Quantitative real-time PCR (qPCR) gene expression measurements of CTR1 were performed on tumor samples obtained after the 22h scan. RESULTS: A relatively high tumor uptake of (64)Cu was seen in four out of five tumor types and an increase in (64)Cu accumulation was seen in three out of five tumor types between 1h and 22h p.i. No relationship was found between tumor uptake of (64)Cu and gene expression of CTR1. CONCLUSIONS: The relatively high, time- and tumor type dependent (64)Cu uptake demonstrated here in five different human cancer xenograft models in mice, emphasizes the importance of validating tracer uptake and indicates that high in vivo stability of copper-based PET tracers is of particular importance because non-tracer-bound copper can accumulate in tumor tissue to a level that could potentially lead to misinterpretation of PET data.
Assuntos
Radioisótopos de Cobre/metabolismo , Imagem Molecular , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Tomografia por Emissão de Pósitrons , Animais , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Transportador de Cobre 1 , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias/patologia , Especificidade de Órgãos , Traçadores RadioativosRESUMO
The correlation between uPAR expression, cancer cell invasion and metastases is now well-established and has prompted the development of a number of uPAR PET imaging agents, which could potentially identify cancer patients with invasive and metastatic lesions. In the present study, we synthesized and characterized two new cross-bridged (64)Cu-labeled peptide conjugates for PET imaging of uPAR and performed a head-to-head comparison with the corresponding and more conventionally used DOTA conjugate. Based on in-source laser-induced reduction of chelated Cu(II) to Cu(I), we now demonstrate the following ranking with respect to the chemical inertness of their complexed Cu ions: DOTA-AE105 << CB-TE2A-AE105 < CB-TE2A-PA-AE105, which is correlated to their corresponding demetallation rate. No penalty in the uPAR receptor binding affinity of the targeting peptide was encountered by conjugation to either of the macrobicyclic chelators (IC50 ~ 5-10 nM) and high yields and radiochemical purities (>95%) were achieved in all cases by incubation at 95ºC. In vivo, they display identical tumor uptake after 1h, but differ significantly after 22 hrs, where the DOTA-AE105 uptake remains surprisingly high. Importantly, the more stable of the new uPAR PET tracers, (64)Cu-CB-TE2A-PA-AE105, exhibits a significantly reduced liver uptake compared to (64)Cu-DOTA-AE105 as well as (64)Cu-CB-TE2A-AE105, (p<0.0001), emphasizing that our new in vitro stability measurements by mass spectrometry predicts in vivo stability in mice. Specificity of the best performing ligand, (64)Cu-CB-TE2A-PA-AE105 was finally confirmed in vivo using a non-binding (64)Cu-labeled peptide as control ((64)Cu-CB-TE2A-PA-AE105(mut)). This control PET-tracer revealed significantly reduced tumor uptake (p<0.0001), but identical hepatic uptake compared to its active counterpart ((64)Cu-CB-TE2A-PA-AE105) after 1h. In conclusion, our new approach using in-source laser-induced reduction of Cu(II)-chelated PET-ligands provides useful information, which are predictive for the tracer stability in vivo in mice. Furthermore, the increased stability of our new macrobicyclic (64)Cu-CB-TE2A-PA-AE105 PET ligand is paralleled by an excellent imaging contrast during non-invasive PET scanning of uPAR expression in preclinical mouse cancer models. The translational promises displayed by this PET-tracer for future clinical cancer patient management remains, however, to be investigated.
Assuntos
Biomarcadores Tumorais/análise , Cobre/metabolismo , Neoplasias/diagnóstico , Peptídeos/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/análise , Coloração e Rotulagem/métodos , Animais , Linhagem Celular Tumoral , Humanos , Ligantes , Camundongos , Camundongos NusRESUMO
UNLABELLED: The proposition of uPAR as a potential target in cancer therapy is advanced by its predominant expression at the invasive front of colorectal cancer (CRC) and its value as prognostic biomarker for poor survival in this disease. In this study, we provide the first in vivo proof-of-concept for a theranostic approach as treatment modality in a human xenograft colorectal cancer model. METHODS: A DOTA-conjugated 9-mer high affinity uPAR binding peptide (DOTA-AE105) was radiolabeled with (64)Cu and (177)Lu, for PET imaging and targeted radionuclide therapy study, respectively. Human uPAR-positive CRC HT-29 cells were inoculated in Nude mice and treated with (177)Lu-DOTA-AE105 once a visible tumor had formed. To evaluate the true effect of the targeted radiotherapy, two controls groups were included in this study, one receiving a (177)Lu-labeled non-binding control peptide and one receiving vehicle. All animals were treated day 0 and 7. A parallel (18)F-FLT PET/CT study was performed on day 0, 1, 3 and 6. Dosimetry calculations were based on a biodistribution study, where organs and tissue of interest were collected 0.5, 1.0, 2.0, 4.0 and 24h post injection of (177)Lu-DOTA-AE105. Toxicity was assessed by recording mouse weight and by H&E staining of kidneys in each treatment group. RESULTS: uPAR-positive HT-29 xenograft was clearly visualized by PET/CT imaging using (64)Cu-DOTA-AE105. Subsequently, these xenograft transplants were locally irradiated using (177)Lu-DOTA-AE105, where a significant effect on tumor size and the number of uPAR-positive cells in the tumor was found (p<0.05). Evaluations of biodistribution and dosimetry revealed highest accumulation of radioactivity in kidneys and tumor tissue. (18)F-FLT PET/CT imaging study revealed a significant correlation between (18)F-FLT tumor uptake and efficacy of the radionuclide therapy. A histological examination of the kidneys from one animal in each treatment group did not reveal any gross abnormalities and the general performance of all treated animals also showed no indications of radioactivity-induced toxicity. CONCLUSION: These findings document for the first time the in vivo efficacy of an uPAR-targeted radionuclide therapeutic intervention on both tumor size and its content of uPAR expressing cells thus setting the stage for future translation into clinical use.
Assuntos
Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Compostos Heterocíclicos com 1 Anel/química , Lutécio/uso terapêutico , Oligopeptídeos/uso terapêutico , Radioisótopos/uso terapêutico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Partículas beta/uso terapêutico , Transformação Celular Neoplásica , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/metabolismo , Didesoxinucleosídeos , Feminino , Células HT29 , Humanos , Camundongos , Terapia de Alvo Molecular , Invasividade Neoplásica , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacocinética , Tomografia por Emissão de Pósitrons , Especificidade por SubstratoRESUMO
INTRODUCTION: The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH(2) labeled with (64)Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) and evaluate their imaging properties using small-animal PET. METHODS: Synthesis of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) was based on solid-phase peptide synthesis protocols using the Fmoc strategy. (68)GaCl(3) was eluted from a (68)Ge/(68)Ga generator. The eluate was either concentrated on a cation-exchange column or fractionated and used directly for labeling. For in vitro characterization of both tracers, partition coefficient, buffer and plasma stability, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft. RESULTS: In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated peptides and identical to AE105. Labeling of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) with (68)Ga was done at 95°C and room temperature, respectively. The highest radiochemical yield and purity were obtained using fractionated elution, whereas a negative effect of acetone on labeling efficiency for NODAGA-AE105-NH(2) was observed. Good stability in phosphate-buffered saline and mouse plasma was observed. High cell uptake was found for both tracers in U87MG tumor cells. Dynamic microPET imaging demonstrated good tumor-to-background ratio for both tracers. Tumor uptake was 2.1% ID/g and 1.3% ID/g 30 min postinjection and 2.0% ID/g and 1.1% ID/g 60 min postinjection for (68)Ga-NODAGA-AE105-NH(2) and (68)Ga-DOTA-AE105-NH(2), respectively. A significantly higher tumor-to-muscle ratio (P<.05) was found for (68)Ga-NODAGA-AE105-NH(2) 60 min postinjection. CONCLUSIONS: The use of (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) as the first gallium-68 labeled uPAR radiotracers for noninvasive PET imaging is reported, which combine versatility with good imaging properties. These new tracers thus constitute an interesting alternative to the (64)Cu-labeled version ((64)Cu-DOTA-AE105 and 64Cu-DOTA-AE105-NH(2)) for detecting uPAR expression in tumor tissue. In our hands, the fractionated elution approach was superior for labeling of peptides, and (68)Ga-NODAGA-AE105-NH(2) is the favored tracer as it provides the highest tumor-to-background ratio.