RESUMO
Facile automatic production is important for the application of prostate-specific membrane antigen (PSMA) tracers in clinical practice. We developed a new 18F-AlF-labelled PSMA probe-18F-AlF-PSMA-NF-and explore its automated production method and potential value in clinical settings. 18F-AlF-PSMA-NF was prepared using an automated method with dimethylformamide (DMF) as the solvent in a positron emission tomography (PET)-MF-2 V-IT-I synthesizer. Tracer characteristics were examined both in vitro and in vivo. Micro-PET/computed tomography (CT) was performed to investigate the utility of 18F-AlF-PSMA-NF for imaging PSMA-positive tumours in vivo. 18F-AlF-PSMA-NF was prepared automatically within 35 min with a non-attenuation correction yield of 37.9 ± 11.2%. The tracer was hydrophilic, had a high affinity for PSMA (Kd = 2.58 ± 0.81 nM), and showed stability in both in vitro and in vivo conditions. In the cellular experiments, 18F-AlF-PSMA-NF uptake in PSMA-positive LNCaP cells was significantly higher than that in PSMA-negative PC-3 cells (P < 0.001), and could be blocked by excess ZJ-43-a PSMA inhibitor (P < 0.001). LNCaP tumours were clearly visualized by 18F-AlF-PSMA-NF on micro-PET/CT, with a high level of uptake (13.72 ± 2.01 percent injected dose per gram of tissue [%ID/g]) and high tumour/muscle ratio (close to 50:1). The PSMA-positive LNCaP tumours had a significantly higher uptake than PSMA-negative PC-3 tumours (13.72 ± 2.01%ID/g vs. 1.07 ± 0.48%ID/g, t = 10.382, P < 0.001), and could be blocked by ZJ-43 (13.72 ± 2.01%ID/g vs. 2.77 ± 1.44%ID/g, t = 8.14, P < 0.001). A new 18F-AlF-labelled PSMA probe-18F-AlF-PSMA-NF-was successfully developed and can be prepared automatically. It has the biological characteristics resembling that of a PSMA-based probe and can potentially be used in clinical settings.
Assuntos
Antígenos de Superfície , Radioisótopos de Flúor , Glutamato Carboxipeptidase II , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Neoplasias da Próstata/diagnóstico por imagem , Compostos Radiofarmacêuticos , Animais , Antígenos de Superfície/química , Antígenos de Superfície/farmacologia , Radioisótopos de Flúor/química , Radioisótopos de Flúor/farmacologia , Glutamato Carboxipeptidase II/síntese química , Glutamato Carboxipeptidase II/química , Glutamato Carboxipeptidase II/farmacocinética , Glutamato Carboxipeptidase II/farmacologia , Humanos , Marcação por Isótopo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células PC-3 , Neoplasias da Próstata/metabolismo , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologia , Distribuição TecidualRESUMO
BACKGROUND: Conventional imaging using CT and bone scan has insufficient sensitivity when staging men with high-risk localised prostate cancer. We aimed to investigate whether novel imaging using prostate-specific membrane antigen (PSMA) PET-CT might improve accuracy and affect management. METHODS: In this multicentre, two-arm, randomised study, we recruited men with biopsy-proven prostate cancer and high-risk features at ten hospitals in Australia. Patients were randomly assigned to conventional imaging with CT and bone scanning or gallium-68 PSMA-11 PET-CT. First-line imaging was done within 21 days following randomisation. Patients crossed over unless three or more distant metastases were identified. The primary outcome was accuracy of first-line imaging for identifying either pelvic nodal or distant-metastatic disease defined by the receiver-operating curve using a predefined reference-standard including histopathology, imaging, and biochemistry at 6-month follow-up. This trial is registered with the Australian New Zealand Clinical Trials Registry, ANZCTR12617000005358. FINDINGS: From March 22, 2017 to Nov 02, 2018, 339 men were assessed for eligibility and 302 men were randomly assigned. 152 (50%) men were randomly assigned to conventional imaging and 150 (50%) to PSMA PET-CT. Of 295 (98%) men with follow-up, 87 (30%) had pelvic nodal or distant metastatic disease. PSMA PET-CT had a 27% (95% CI 23-31) greater accuracy than that of conventional imaging (92% [88-95] vs 65% [60-69]; p<0·0001). We found a lower sensitivity (38% [24-52] vs 85% [74-96]) and specificity (91% [85-97] vs 98% [95-100]) for conventional imaging compared with PSMA PET-CT. Subgroup analyses also showed the superiority of PSMA PET-CT (area under the curve of the receiver operating characteristic curve 91% vs 59% [32% absolute difference; 28-35] for patients with pelvic nodal metastases, and 95% vs 74% [22% absolute difference; 18-26] for patients with distant metastases). First-line conventional imaging conferred management change less frequently (23 [15%] men [10-22] vs 41 [28%] men [21-36]; p=0·008) and had more equivocal findings (23% [17-31] vs 7% [4-13]) than PSMA PET-CT did. Radiation exposure was 10·9 mSv (95% CI 9·8-12·0) higher for conventional imaging than for PSMA PET-CT (19·2 mSv vs 8·4 mSv; p<0·001). We found high reporter agreement for PSMA PET-CT (κ=0·87 for nodal and κ=0·88 for distant metastases). In patients who underwent second-line image, management change occurred in seven (5%) of 136 patients following conventional imaging, and in 39 (27%) of 146 following PSMA PET-CT. INTERPRETATION: PSMA PET-CT is a suitable replacement for conventional imaging, providing superior accuracy, to the combined findings of CT and bone scanning. FUNDING: Movember and Prostate Cancer Foundation of Australia. VIDEO ABSTRACT.
Assuntos
Antígenos de Superfície/administração & dosagem , Glutamato Carboxipeptidase II/administração & dosagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias da Próstata/diagnóstico , Imagem Corporal Total/métodos , Idoso , Antígenos de Superfície/farmacologia , Biomarcadores , Glutamato Carboxipeptidase II/farmacologia , Humanos , Metástase Linfática/diagnóstico por imagem , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/diagnóstico por imagem , Estudos Prospectivos , Neoplasias da Próstata/patologia , Sensibilidade e EspecificidadeRESUMO
Prostate-specific membrane antigen (PSMA)-targeted radioligands have been used for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Recently, albumin-binding PSMA radioligands with enhanced blood circulation were developed to increase the tumor accumulation of activity. The present study aimed at the design, synthesis and preclinical evaluation of a novel class of PSMA-targeting radioligands equipped with ibuprofen as a weak albumin-binding entity in order to improve the pharmacokinetic properties. Methods: Four novel glutamate-urea-based PSMA ligands were synthesized with ibuprofen, conjugated via variable amino acid-based linker entities. The albumin-binding properties of the 177Lu-labeled PSMA ligands were tested in vitro using mouse and human plasma. Affinity of the radioligands to PSMA and cellular uptake and internalization was investigated using PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu tumor cells. The tissue distribution profile of the radioligands was assessed in biodistribution and imaging studies using PC-3 PIP/flu tumor-bearing nude mice. Results: The PSMA ligands were obtained in moderate yields at high purity (>99%). 177Lu-labeling of the ligands was achieved at up to 100 MBq/nmol with >96% radiochemical purity. In vitro assays confirmed high binding of all radioligands to mouse and human plasma proteins and specific uptake and internalization into PSMA-positive PC-3 PIP tumor cells. Biodistribution studies and SPECT/CT scans revealed high accumulation in PC-3 PIP tumors but negligible uptake in PC-3 flu tumor xenografts as well as rapid clearance of activity from background organs and tissues. 177Lu-Ibu-DAB-PSMA, in which ibuprofen was conjugated via a positively-charged diaminobutyric acid (DAB) entity, showed distinguished tumor uptake and the most favorable tumor-to-blood and tumor-to-kidney ratios. Conclusion: The high accumulation of activity in the tumor and fast clearance from background organs was a common favorable characteristic of PSMA radioligands modified with ibuprofen as albumin-binding entity. 177Lu-Ibu-DAB-PSMA emerged as the most promising candidate; hence, more detailed preclinical investigations with this radioligand are warranted in view of a clinical translation.
Assuntos
Albuminas/metabolismo , Antígenos de Superfície/farmacologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Glutamato Carboxipeptidase II/farmacologia , Ibuprofeno/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/secundário , Animais , Antígenos de Superfície/administração & dosagem , Antígenos de Superfície/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacocinética , Feminino , Glutamato Carboxipeptidase II/administração & dosagem , Glutamato Carboxipeptidase II/metabolismo , Humanos , Ibuprofeno/farmacocinética , Injeções Subcutâneas , Ligantes , Lutécio/metabolismo , Masculino , Camundongos , Camundongos Nus , Radioisótopos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Albumina Sérica Humana , Soroglobulinas , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto/estatística & dados numéricosRESUMO
BACKGROUND: Prostate cancer (PCa) is the second leading cause of cancer-related death in the Western population. The use in oncology of positron emission tomography/computed tomography (PET/CT) with emerging radiopharmaceuticals promises accurate staging of primary disease, restaging of recurrent disease and detection of metastatic lesions. Prostate-specific membrane antigen (PSMA) expression, directly related to androgen-independence, metastasis and progression, renders this tumour associate antigen a good target for the development of new radiopharmaceuticals for PET. Aim of this study was to demonstrate in a preclinical in vivo model (PSMA-positive versus PSMA-negative tumours) the targeting specificity and sensitivity of the anti-PSMA single-chain variable fragment (scFv) labelled with 124I. METHODS: The 124I-labeling conditions of the antibody fragment scFvD2B were optimized and assessed for purity and immunoreactivity. The specificity of 124I-scFvD2B was tested in mice bearing PSMA-positive and PSMA-negative tumours to assess both ex-vivo biodistribution and immune-PET. RESULTS: The uptake fraction of 124I-scFvD2B was very high on PSMA positive cells (range 75-91%) and highly specific and immuno-PET at the optimal time point, defined between 15 h and 24 h, provides a specific localization of lesions bearing the target antigen of interest (PSMA positive vs PSMA negative tumors %ID/g: p = 0.0198 and p = 0.0176 respectively) yielding a median target/background ratio around 30-40. CONCLUSIONS: Preclinical in vivo results of our immuno-PET reagent are highly promising. The target to background ratio is improved notably using PET compared to SPECT previously performed. These data suggest that, upon clinical confirmation of sensitivity and specificity, our anti-PSMA 124I-scFvD2B may be superior to other diagnostic modalities for PCa. The possibility to combine in patients our 124I-scFvD2B in multi-modal systems, such as PET/CT, PET/MR and PET/SPECT/CT, will provide quantitative 3D tomographic images improving the knowledge of cancer biology and treatment.
Assuntos
Antígenos de Superfície/imunologia , Glutamato Carboxipeptidase II/imunologia , Neoplasias da Próstata/diagnóstico , Compostos Radiofarmacêuticos/farmacologia , Anticorpos de Cadeia Única/imunologia , Animais , Antígenos de Superfície/farmacologia , Linhagem Celular Tumoral , Glutamato Carboxipeptidase II/farmacologia , Humanos , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Radioisótopos do Iodo/farmacologia , Masculino , Camundongos , Metástase Neoplásica , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Compostos Radiofarmacêuticos/imunologia , Anticorpos de Cadeia Única/farmacologia , Distribuição TecidualRESUMO
OBJECTIVES: To determine the diagnostic accuracy of 68gallium prostate-specific membrane antigen (PSMA)-based positron emission tomography/computed tomography (PET/CT) in comparison with 18F-fluoride-based PET/CT (NaF-PET/CT) and whole-body magnetic resonance imaging (WB-MRI) for the detection of bone metastases in patients with prostate cancer. METHODS: Sixty patients with prostate cancer were included in the period May 2016 to June 2017. The participants underwent three scans (index tests) within 30 days: a NaF-PET/CT, a WB-MRI and a PSMA-PET/CT. Experienced specialists assessed the scans. In the absence of a histological reference standard, the final diagnosis was determined as a panel diagnosis. Measures of the diagnostic performances of the index tests were calculated from patient-based dichotomous outcomes (0 or ≥ 1 bone metastasis) and pairwise compared (McNemar test). For each index test, the agreement with the final diagnosis with regard to the number of bone metastases detected (0, 1-5, > 5) and the inter-reader agreement was calculated (kappa coefficients). RESULTS: Fifty-five patients constituted the final study population; 20 patients (36%) were classified as having bone metastatic disease as their final diagnosis. The patient-based diagnostic performances were (sensitivity, specificity, overall accuracy) PSMA-PET/CT (100%, 100%, 100%), NaF-PET/CT (95%, 97%, 96%) and WB-MRI (80%, 83%, 82%). The overall accuracy of PSMA-PET/CT was significantly more favourable compared to WB-MRI (p = 0.004), but not to NaF-PET/CT (p = 0.48). PSMA-PET/CT classified the number of bone metastases reliably compared to the final diagnosis (kappa coefficient 0.97) and with an "almost perfect" inter-reader agreement (kappa coefficient 0.93). CONCLUSIONS: The overall accuracy of PSMA-PET/CT was significantly more advantageous compared to WB-MRI, but not to NaF-PET/CT. KEY POINTS: ⢠PSMA-PET/CT assessed the presence of bone metastases correctly in all 55 patients ⢠PSMA-PET/CT was more advantageous compared to WB-MRI ⢠No difference was found between PSMA-PET/CT and NaF-PET/CT.
Assuntos
Antígenos de Superfície/farmacologia , Neoplasias Ósseas/secundário , Radioisótopos de Gálio/farmacologia , Glutamato Carboxipeptidase II/farmacologia , Imageamento por Ressonância Magnética/métodos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias da Próstata/patologia , Imagem Corporal Total/métodos , Idoso , Idoso de 80 Anos ou mais , Neoplasias Ósseas/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Reprodutibilidade dos TestesRESUMO
Prostate-specific membrane antigen (PSMA) PET has been recently introduced for the diagnosis of patients with metastatic prostate cancer (PCa). Until today, staging of patients with PCa relied mostly on morphologic features, such as size or shape, resulting in low detection rates in disease recurrence. PSMA PET imaging provides molecular information and, in combination with conventional imaging, offers improved sensitivity and specificity. This review discusses the benefits and limitations of PSMA imaging in the setting of primary staging and detection of recurrent disease in comparison with standard-of-care imaging techniques.
Assuntos
Antígenos de Superfície/farmacologia , Glutamato Carboxipeptidase II/farmacologia , Estadiamento de Neoplasias/métodos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias da Próstata/diagnóstico , Humanos , Masculino , Período Pré-Operatório , Compostos Radiofarmacêuticos/farmacologiaRESUMO
Prostate-specific membrane antigen (PSMA) is a membrane protein that is overexpressed manifold in prostate cancer and provides an attractive target for molecular therapy. Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photoabsorber conjugate (APC). Here, we describe the efficacy of NIR-PIT, using a fully human IgG1 anti-PSMA monoclonal antibody (mAb), conjugated to the photoabsorber, IR700DX, in a PSMA-expressing PC3 prostate cancer cell line. Anti-PSMA-IR700 showed specific binding and cell-specific killing was observed after exposure of the cells to NIR light in vitro In the in vivo study, anti-PSMA-IR700 showed high tumor accumulation and high tumor-background ratio. Tumor-bearing mice were separated into 4 groups: (i) no treatment; (ii) 100 µg of anti-PSMA-IR700 i.v.; (iii) NIR light exposure; (iv) 100 µg of anti-PSMA-IR700 i.v., NIR light exposure was administered. These were performed every week for up to 3 weeks. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other control groups (P < 0.001), and significantly prolonged survival was achieved (P < 0.0001 vs. other control groups). More than two thirds of tumors were cured with NIR-PIT. In conclusion, the anti-PSMA antibody is suitable as an APC for NIR-PIT. Furthermore, NIR-PIT with the anti-PSMA-IR700 antibody is a promising candidate of the treatment of PSMA-expressing tumors and could be readily translated to humans.Implications: NIR-infrared photoimmunotherapy (NIR-PIT) using a fully human anti-PSMA-IR700 conjugate showed potential therapeutic effects against a PSMA-expressing prostate cancer that is readily translated to humans. Mol Cancer Res; 15(9); 1153-62. ©2017 AACR.
Assuntos
Antígenos de Superfície/uso terapêutico , Glutamato Carboxipeptidase II/uso terapêutico , Imunoterapia/métodos , Fototerapia/métodos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/terapia , Animais , Antígenos de Superfície/farmacologia , Linhagem Celular Tumoral , Feminino , Glutamato Carboxipeptidase II/farmacologia , Humanos , Masculino , Camundongos , Camundongos NusRESUMO
OBJECTIVE: To assess the diagnostic accuracy of 64Copper prostate-specific membrane antigen (64Cu-PSMA) positron emission tomography/computed tomography (PET/CT) in the primary lymph node (LN) staging of a selected cohort of intermediate- to high-risk prostate cancer (PCa) patients. MATERIALS AND METHODS: An observational prospective study was performed in 23 patients with intermediate- to high-risk PCa, who underwent 64Cu-PSMA PET/CT for local and lymph nodal staging before laparoscopic radical prostatectomy with an extended pelvic LN dissection. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for LN status of 64Cu-PSMA PET/CT were calculated using the final pathological findings as reference. Furthermore, we evaluated the correlation of intraprostatic tumor extent and grading with 64Cu-PSMA intraprostatic distribution. RESULTS: Pathological analysis of LN involvement in 413 LNs harvested from our study cohort identified a total of 22 LN metastases in 8 (5%) of the 23 (35%) PCa patients. Imaging-based LN staging in a per-patient analysis showed that 64Cu-PSMA PET/CT was positive in 7 of 8 LN-positive patients (22%) with a sensitivity of 87.5%, specificity of 100%, PPV of 100%, and NPV of 93.7%, considering the maximum standardized uptake value (SUVmax) at 4 hours as our reference. Receiver operating characteristic curve was characterized by an area under the curve of 0.938. A significant positive association was observed between SUVmax at 4 hours with Gleason score, index, and cumulative tumor volume. CONCLUSION: In our intermediate- to high-risk PCa patients study cohort, we showed the high diagnostic accuracy of 64Cu-PSMA PET/CT for primary LN staging before radical prostatectomy.
Assuntos
Antígenos de Superfície/farmacologia , Radioisótopos de Cobre/farmacologia , Glutamato Carboxipeptidase II/farmacologia , Linfonodos/diagnóstico por imagem , Estadiamento de Neoplasias/métodos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias da Próstata/diagnóstico , Idoso , Humanos , Laparoscopia , Metástase Linfática , Masculino , Estudos Prospectivos , Prostatectomia/métodos , Neoplasias da Próstata/secundário , Neoplasias da Próstata/cirurgia , Curva ROC , Reprodutibilidade dos Testes , Fatores de RiscoRESUMO
Docetaxel (DOC) is used for the first-line treatment of castration resistant prostate cancer (CPRC). However, the therapeutic effects are limited, only about one half of patients respond to the therapy and severe side effects possibly lead to discontinuation of treatment. Therefore, actual research is focused on the development of new DOC-based combination treatments. In this study we investigated the antitumor effects of a recombinant immunotoxin targeting the prostate specific membrane antigen (PSMA) in combination with DOC in vitro and in vivo. The immunotoxin consists of an anti-PSMA single chain antibody fragment (scFv) as binding and a truncated form of Pseudomonas aeruginosa Exotoxin A (PE40) as toxin domain. The immunotoxin induced apoptosis and specifically reduced the viability of androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cells. A synergistic cytotoxic activity was observed in combination with DOC with IC50 values in the low picomolar or even femtomolar range. Moreover, combination treatment resulted in an enhanced antitumor activity in a C4-2 SCID mouse xenograft model. This highlights the immunotoxin as a promising therapeutic agent for a future DOC-based combination therapy of CPRC.
Assuntos
Antígenos de Superfície/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Glutamato Carboxipeptidase II/farmacologia , Imunotoxinas/farmacologia , Neoplasias da Próstata , Animais , Linhagem Celular Tumoral , Docetaxel , Humanos , Masculino , Camundongos , Camundongos SCID , Anticorpos de Cadeia Única/farmacologia , Taxoides/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Reports remain insufficient on whether and how prostate-specific membrane antigen (PSMA) can influence in vivo osseous metastasis of prostate cancer (PCa). In the present study, the authors induced stable expression of PSMA in mouse PCa cell line RM-1. In vivo osseous metastasis was induced in 37 6-week-old female C57BL/6 mice weighing 22.45 ± 0.456 g. RM-1 cells were actively injected into the femoral bone cavity, leading to bilateral dissymmetry of bone density in the femoral bone. Tumor cells were also detected in bone tissue by pathological examination. The impact on bone density was demonstrated by the significant difference between animals injected with RM-PSMA cells (0.0738 ± 0.0185 g/cm²) and animals injected with RM-empty plasmid cells (0.0895 ± 0.0241 g/cm²). The lytic bone lesion of the RM-PSMA group (68.4%) was higher than that of the control group (27.8%). Immunohistochemistry showed that the expression of both vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) was distinctly higher in the RM-PSMA group than in the control group, while ELISA and Western blot assay indicated that VEGF and MMP-9 were higher in the RM-PSMA group compared to the control group (in vitro). Thus, the present study proposed and then confirmed for the first time that PSMA can promote in vivo osseous metastasis of PCa by increasing sclerotic destruction of PCa cells. Further analyses also suggested that PSMA functions positively on the invasive ability of RM-1 by increasing the expression of MMP-9 and VEGF by osseous metastases in vivo.
Assuntos
Animais , Feminino , Masculino , Camundongos , Antígenos de Superfície/metabolismo , Neoplasias Ósseas/secundário , Glutamato Carboxipeptidase II/metabolismo , Neoplasias da Próstata/patologia , Antígenos de Superfície/farmacologia , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/fisiologia , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Glutamato Carboxipeptidase II/farmacologia , Imuno-Histoquímica , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias da Próstata/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Reports remain insufficient on whether and how prostate-specific membrane antigen (PSMA) can influence in vivo osseous metastasis of prostate cancer (PCa). In the present study, the authors induced stable expression of PSMA in mouse PCa cell line RM-1. In vivo osseous metastasis was induced in 37 6-week-old female C57BL/6 mice weighing 22.45 ± 0.456 g. RM-1 cells were actively injected into the femoral bone cavity, leading to bilateral dissymmetry of bone density in the femoral bone. Tumor cells were also detected in bone tissue by pathological examination. The impact on bone density was demonstrated by the significant difference between animals injected with RM-PSMA cells (0.0738 ± 0.0185 g/cm²) and animals injected with RM-empty plasmid cells (0.0895 ± 0.0241 g/cm²). The lytic bone lesion of the RM-PSMA group (68.4%) was higher than that of the control group (27.8%). Immunohistochemistry showed that the expression of both vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) was distinctly higher in the RM-PSMA group than in the control group, while ELISA and Western blot assay indicated that VEGF and MMP-9 were higher in the RM-PSMA group compared to the control group (in vitro). Thus, the present study proposed and then confirmed for the first time that PSMA can promote in vivo osseous metastasis of PCa by increasing sclerotic destruction of PCa cells. Further analyses also suggested that PSMA functions positively on the invasive ability of RM-1 by increasing the expression of MMP-9 and VEGF by osseous metastases in vivo.
Assuntos
Antígenos de Superfície/metabolismo , Neoplasias Ósseas/secundário , Glutamato Carboxipeptidase II/metabolismo , Neoplasias da Próstata/patologia , Animais , Antígenos de Superfície/farmacologia , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/fisiologia , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Feminino , Glutamato Carboxipeptidase II/farmacologia , Imuno-Histoquímica , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias da Próstata/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Gene directed enzyme pro-drug therapy (GDEPT) is one of the adjuvant therapeutic regimens for advanced prostate adenocarcinoma, and this research intended to explore how to apply targeting therapy of prostate adenocarcinoma under the mediation of a promoter/enhancer of prostate-specific membrane antigen (PSMA(EP)) as a specific regulatory element. Recombinant adenoviruses (Ad-PSMA(E-P)-enhanced green fluorescent protein [EGFP], Ad-CMV-EGFP, Ad-PSMA(E-P)-CD, and Ad-CMV-CD) were constructed and could express cytosine deaminase (CD) or the EGFP reporter gene driven by a PSMA(EP) or cytomegalovirus (CMV) promoter. LNCaP, CL-1, MCF-7, and A549 were infected with CD-produced recombinant adenoviruses and treated with pro-drug 5-fluorocytosine (5-FC) in vivo and vitro; then, the growth inhibition of the cells and the cell cycle variation were assessed by an [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay and flow cytometry. Growth suppression of the xenograft tumor was also adopted to evaluate the efficiency of the suicide system. Morphologic changes after treatment in vivo were assessed with hematoxylin and eosin staining. In the 4 examined cancer cell lines, PSMA-positive prostate cancer cells LNCap and CL-1 were exclusively sensitive to the Ad-PSMA(E-P)-CD/5-FC system. The S phase of cell cycle arrest was thought to be involved in the cytotoxicity of 5-fluorouracil (5-FU) converted from 5-FC by CD. CL-1 implanted Athymic BALB/c mice showed growth inhibition of tumors when they were treated with the Ad-PSMA(E-P)-CD/5-FC system without systemic conversion toxicity. The PSMA-based, CD-produced adenovirus, deserving further investigation in the future, might be a good candidate for targeting gene therapy of prostate adenocarcinoma.
Assuntos
Adenoviridae/genética , Antígenos de Superfície/genética , Terapia Genética , Glutamato Carboxipeptidase II/genética , Antígenos de Superfície/farmacologia , Antígenos de Superfície/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citomegalovirus/genética , Elementos Facilitadores Genéticos , Genes Reporter , Glutamato Carboxipeptidase II/farmacologia , Glutamato Carboxipeptidase II/toxicidade , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Plasmídeos , Regiões Promotoras Genéticas , Neoplasias da Próstata , Recombinação Genética , Transcrição GênicaRESUMO
Prostate cancer is the most commonly diagnosed form of cancer and the second leading cancer-related death among men in the Western civilization. Since no effective therapy exists for this tumor after progression beyond resectable boundaries, there is an urgent need for new treatment strategies. Prostate specific membrane antigen (PSMA) represents an excellent target on prostate cancer cells, and therefore specific immunotherapy may be a novel therapeutic option for the management of this tumor. We constructed a fully recombinant immunotoxin (A5-PE40) from a single-chain antibody fragment (scFv) against cell-adherent PSMA and a truncated form of Pseudomonas exotoxin A (PE40) lacking its natural binding domain Ia. The scFv A5 was obtained from a mAb elicited with native PSMA by phage display technology and direct selection on cells carrying the antigen. The bacterially expressed and purified immunotoxin A5-PE40 specifically binds to PSMA-positive prostate cancer cells and induces a 50% reduction of viability (IC50) at a concentration of 20 pM, while PSMA-negative cells remain unaffected. Due to its high and specific toxicity this recombinant immunotoxin is a promising candidate for therapeutic applications in patients with prostate cancer.