RESUMO
We investigated the potential use of [18F]FDG PET as a response biomarker for PI3K pathway targeting therapies in two HER-2-overexpressing cancer models. Methods. CD-1 nude mice were inoculated with HER-2-overexpressing JIMT1 (trastuzumab-resistant) or SKOV3 (trastuzumab-sensitive) human cancer cells. Animals were treated with trastuzumab, everolimus (mTOR inhibitor), PIK90 (PI3K inhibitor), saline, or combination therapy. [18F]FDG scans were performed at baseline, two, and seven days after the start of the therapy. Tumors were delineated on CT images and relative tumor volumes (RTV) and maximum standardized uptake value (SUVmax) were calculated. Levels of pS6 and pAkt on protein tumor lysates were determined with ELISA. Results. In the SKOV3 xenografts, all treatment schedules resulted in a gradual decrease in RTV and delta SUVmax (ΔSUVmax). For all treatments combined, ΔSUVmax after 2 days was predictive for RTV after 7 days (r = 0.69, p = 0.030). In JIMT1 tumors, monotherapy with everolimus or PIK90 resulted in a decrease in RTV (-30% ± 10% and -20% ± 20%, respectively) and ΔSUVmax (-39% ± 36% and -42% ± 8%, respectively) after 7 days of treatment, but not earlier, while trastuzumab resulted in nonsignificant increases compared to control. Combination therapies resulted in RTV and ΔSUVmax decrease already at day 2, except for trastuzumab+everolimus, where an early flare was observed. For all treatments combined, ΔSUVmax after 2 days was predictive for RTV after 7 days (r = 0.48, p = 0.028), but the correlation could be improved when combination with everolimus (r = 0.59, p = 0.023) or trastuzumab (r = 0.69, p = 0.015) was excluded. Conclusion. Reduction in [18F]FDG after 2 days correlated with tumor volume changes after 7 days of treatment and confirms the use of [18F]FDG PET as an early response biomarker. Treatment response can however be underestimated in schedules containing trastuzumab or everolimus due to temporary increased [18F]FDG uptake secondary to negative feedback loop and crosstalk between different pathways.
Assuntos
Neoplasias , Preparações Farmacêuticas , Animais , Fluordesoxiglucose F18 , Xenoenxertos , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Serina-Treonina Quinases TORRESUMO
Bone metastases remain a common feature of advanced cancers and are associated with significant morbidity and mortality. Recent research has identified promising novel treatment targets to improve current treatment strategies for bone metastatic disease. This review summarizes the well-known and recently discovered molecular biology pathways in bone that govern normal physiological remodeling or drive the pathophysiological changes observed when bone metastases are present. In the rapidly changing world of targeted cancer treatments, it is important to recognize the specific treatment effects induced in bone by these agents and the potential impact on common imaging strategies. The osteoclastic targets (bisphosphonates, LGR4, RANKL, mTOR, MET-VEGFR, cathepsin K, Src, Dock 5) and the osteoblastic targets (Wnt and endothelin) are discussed, and the emerging field of osteo-immunity is introduced as potential future therapeutic target. Finally, a summary is provided of available trial data for agents that target these pathways and that have been assessed in patients. The ultimate goal of research into novel pathways and targets involved in the tumor-bone microenvironment is to tackle one of the great remaining unmet needs in oncology, that is finding a cure for bone metastatic disease.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Terapia de Alvo Molecular/métodos , Animais , Matriz Óssea/efeitos dos fármacos , Matriz Óssea/metabolismo , Matriz Óssea/patologia , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/patologia , Humanos , Imunidade Inata/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
MMP-9 is a zinc-dependent endopeptidase that is involved in the proteolytic degradation of the extracellular matrix and plays an important role in cancer migration, invasion, and metastasis. The aim of this study was to evaluate the potential of MMP-tracers [18 F]BR420 and [18 F]BR351 for MMP-9 imaging in a colorectal cancer xenograft model. [18 F]BR420 and [18 F]BR351 were synthesized using an automated synthesis module. For [18 F]BR420, a novel and improved radiosynthesis was developed. Plasma stability and MMP-9-targeting capacity of both radiotracers was compared in the Colo205 colorectal cancer model. MMP-9 and MMP-2 expression levels in the tumors were evaluated by immunohistochemistry and in situ zymography. µPET imaging as well as ex vivo biodistribution revealed a higher tumor uptake for [18 F]BR420 (3.15% ± 0.03% ID/g vs 0.94% ± 0.18% ID/g for [18 F]BR351 at 2 hours pi) but slower blood clearance compared with [18 F]BR351. [18 F]BR351 was quickly metabolized in plasma with 20.28% ± 5.41% of intact tracer remaining at 15 minutes postinjection (PI). By contrast, [18 F]BR420 displayed a higher metabolic stability with >86% intact tracer remaining at 2 hours PI. Immunohistochemistry revealed the presence of MMP-9 and MMP-2 in the tumor tissue, which was confirmed by in situ zymography. However, an autoradiography analysis of tracer distribution in the tumors did not correlate with MMP-9 expression. [18 F]BR420 displayed a higher tumor uptake and higher stability compared with [18 F]BR351 but a low tumor-to-blood ratio and discrepancy between tracer distribution and MMP-9 immunohistochemistry. Therefore, both tracers will not be usefulness for MMP-9 imaging in colorectal cancer.
Assuntos
Neoplasias Colorretais/diagnóstico por imagem , Metaloproteinase 9 da Matriz/metabolismo , Pirimidinonas/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Sulfonamidas/síntese química , Valina/análogos & derivados , Animais , Linhagem Celular Tumoral , Feminino , Radioisótopos de Flúor/química , Humanos , Taxa de Depuração Metabólica , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Pirimidinonas/farmacocinética , Compostos Radiofarmacêuticos/efeitos adversos , Compostos Radiofarmacêuticos/síntese química , Sulfonamidas/farmacocinética , Distribuição Tecidual , Valina/síntese química , Valina/farmacocinéticaRESUMO
BACKGROUND: The mTOR-inhibitor everolimus improves progression-free survival in advanced pancreatic neuroendocrine tumours (PNETs). However, adaptive resistance to mTOR inhibition is described. METHODS: QGP-1 and BON-1, two human PNET cell lines, were cultured with increasing concentrations of everolimus up to 22 weeks to reach a dose of 1 µM everolimus, respectively, 1000-fold and 250-fold initial IC50. Using total DNA content as a measure of cell number, growth inhibitory dose-response curves of everolimus were determined at the end of resistance induction and over time after everolimus withdrawal. Response to ATP-competitive mTOR inhibitors OSI-027 and AZD2014, and PI3K-mTOR inhibitor NVP-BEZ235 was studied. Gene expression of 10 PI3K-Akt-mTOR pathway-related genes was evaluated using quantitative real-time PCR (RT-qPCR). RESULTS: Long-term everolimus-treated BON-1/R and QGP-1/R showed a significant reduction in everolimus sensitivity. During a drug holiday, gradual return of everolimus sensitivity in BON-1/R and QGP-1/R led to complete reversal of resistance after 10-12 weeks. Treatment with AZD2014, OSI-027 and NVP-BEZ235 had an inhibitory effect on cell proliferation in both sensitive and resistant cell lines. Gene expression in BON-1/R revealed downregulation of MTOR, RICTOR, RAPTOR, AKT and HIF1A, whereas 4EBP1 was upregulated. In QGP-1/R, a downregulation of HIF1A and an upregulation of ERK2 were observed. CONCLUSIONS: Long-term everolimus resistance was induced in two human PNET cell lines. Novel PI3K-AKT-mTOR pathway-targeting drugs can overcome everolimus resistance. Differential gene expression profiles suggest different mechanisms of everolimus resistance in BON-1 and QGP-1.
Assuntos
Everolimo/farmacologia , Tumores Neuroendócrinos/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Tumores Neuroendócrinos/enzimologia , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Triazinas/farmacologiaRESUMO
Recently, bioorthogonal chemistry based on the Inverse Electron-Demand Diels-Alder (IEDDA) cycloaddition between 1,2,4,5-tetrazines and trans-cyclooctene (TCO) analogues added an interesting dimension to molecular imaging. Until now, antibodies (Abs) were tagged with TCO and after pretargeting they were reacted with tetrazines substituted with reporters. However, TCO tags have the tendency to degrade under physiological conditions, and due to their hydrophobic nature are buried within the protein. This results in loss of reactivity and a low Ab functional loading. To circumvent these problems, we report for the first time an approach in which tetrazines are used as tags for antibody (Ab) modification, and TCO as the imaging agent. We developed a new Ab-tetrazine conjugate, which displays a high functional loading, good stability and reactivity. We utilized this immunoconjugate for live-cell imaging together with novel TCO probes, resulting in selective and rapid labeling of SKOV-3 cells. Our approach may be useful for in vivo pretargeted imaging.
RESUMO
BACKGROUND: The cysteine-aspartic acid protease caspase-3 is recognized as the main executioner of apoptosis in cells responding to specific extrinsic and intrinsic stimuli. Caspase-3 represents an interesting biomarker to evaluate treatment response, as many cancer therapies exert their effect by inducing tumour cell death. Previously developed caspase-3 PET tracers were unable to reach routine clinical use due to low tumour uptake or lack of target selectivity, which are two important requirements for effective treatment response evaluation in cancer patients. Therefore, the goal of this study was to develop and preclinically evaluate novel caspase-3-selective activity-based probes (ABPs) for apoptosis imaging. RESULTS: A library of caspase-3-selective ABPs was developed for tumour apoptosis detection. In a first attempt, the inhibitor Ac-DW3-KE (Ac-3Pal-Asp-ßhLeu-Phe-Asp-KE) was 18F-labelled on the N-terminus to generate a radiotracer that was incapable of adequately detecting an increase in apoptosis in vivo. The inability to effectively detect active caspase-3 in vivo was likely attributable to slow binding, as demonstrated with in vitro inhibition kinetics. Hence, a second generation of caspase-3 selective ABPs was developed based on the Ac-ATS010-KE (Ac-3Pal-Asp-Phe(F5)-Phe-Asp-KE) with greatly improved binding kinetics over Ac-DW3-KE. Our probes based on Ac-ATS010-KE were made by modifying the N-terminus with 6 different linkers. All the linker modifications had limited effect on the binding kinetics, target selectivity, and pharmacokinetic profile in healthy mice. In an in vitro apoptosis model, the least hydrophilic tracer [18F]MICA-316 showed an increased uptake in apoptotic cells in comparison to the control group. Finally, [18F]MICA-316 was tested in an in vivo colorectal cancer model, where it showed a limited tumour uptake and was unable to discriminate treated tumours from the untreated group, despite demonstrating that the radiotracer was able to bind caspase-3 in complex mixtures in vitro. In contrast, the phosphatidylethanolamine (PE)-binding radiotracer [99mTc]Tc-duramycin was able to recognize the increased cell death in the disease model, making it the best performing treatment response assessment tracer developed thus far. CONCLUSIONS: In conclusion, a novel library of caspase-3-binding PET tracers retaining similar binding kinetics as the original inhibitor was developed. The most promising tracer, [18F]MICA-316, showed an increase uptake in an in vitro apoptosis model and was able to selectively bind caspase-3 in apoptotic tumour cells. In order to distinguish therapy-responsive from non-responsive tumours, the next generation of caspase-3-selective ABPs will be developed with higher tumour accumulation and in vivo stability.
RESUMO
Tumor hypoxia and tumor metabolism are linked through the activation of metabolic genes following hypoxia-inducible factor 1 (HIF-1) activation. This raises the question of whether this relationship can be exploited to improve 2-deoxy-2-[(18)F]fluoro-D-glucose positron emission tomography ([(18)F]FDG-PET). To do this, [(18)F]FDG uptake was investigated after chemical induction of hypoxia and chemical activation of HIF-1 in an in vitro and an in vivo model of a human colorectal carcinoma. [(18)F]FDG uptake, HIF-1α protein levels, and messenger ribonucleic acid expression of glucose transporter 1 (GLUT1), hexokinase 2, HIF-1α, and carbonic anhydrase IX (CA IX) were determined in HT29 cells after treatment with 200 µM CoCl(2) and 500 µM dimethyloxalylglycine (DMOG). In an HT29 xenograft, the distribution of endogenous and exogenous markers of hypoxia was investigated using immunohistochemistry, and tumor [(18)F]FDG uptake was determined after treatment with a single dose of 5 mg/kg hydralazine and 8 mg DMOG. Treatment of HT29 cells with CoCl(2) and DMOG induced functional HIF-1 and resulted in increased [(18)F]FDG uptake. In an HT29 xenograft, a similar spatial distribution of pimonidazole, CA IX, and GLUT1 was found, and treatment with DMOG resulted in significant increases in maximum and mean standardized uptake values using [(18)F]FDG-PET. Chemical activation of HIF-1 can increase in vitro and in vivo [(18)F]FDG uptake. Imaging after pharmacologic HIF-1 activation might increase tumor [(18)F]FDG uptake when using [(18)F]FDG-PET.
Assuntos
Hipóxia Celular/fisiologia , Fluordesoxiglucose F18/farmacocinética , Neoplasias Experimentais/metabolismo , Aminoácidos Dicarboxílicos , Análise de Variância , Animais , Encéfalo/metabolismo , Química Encefálica , Cobalto , Feminino , Células HT29 , Humanos , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Nus , Imagem Molecular/métodos , Neoplasias Experimentais/química , Neoplasias Experimentais/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background: The pretargeted imaging strategy using inverse electron demand Diels-Alder (IEDDA) cycloaddition between a trans-cyclooctene (TCO) and tetrazine (Tz) has emerged and rapidly grown as a promising concept to improve radionuclide imaging and therapy in oncology. This strategy has mostly relied on the use of radiolabeled Tz together with TCO-modified targeting vectors leading to a rapid growth of the number of available radiolabeled tetrazines, while only a few radiolabeled TCOs are currently reported. Here, we aim to develop novel and structurally diverse 18F-labeled cis-dioxolane-fused TCO (d-TCO) derivatives to further expand the bioorthogonal toolbox for in vivo ligation and evaluate their potential for positron emission tomography (PET) pretargeted imaging. Results: A small series of d-TCO derivatives were synthesized and tested for their reactivity against tetrazines, with all compounds showing fast reaction kinetics with tetrazines. A fluorescence-based pretargeted blocking study was developed to investigate the in vivo ligation of these compounds without labor-intensive prior radiochemical development. Two compounds showed excellent in vivo ligation results with blocking efficiencies of 95 and 97%. Two novel 18F-labeled d-TCO radiotracers were developed, from which [18F]MICA-214 showed good in vitro stability, favorable pharmacokinetics, and moderate in vivo stability. Micro-PET pretargeted imaging with [18F]MICA-214 in mice bearing LS174T tumors treated with tetrazine-modified CC49 monoclonal antibody (mAb) (CC49-Tz) showed significantly higher uptake in tumor tissue in the pretargeted group (CC49-Tz 2.16 ± 0.08% ID/mL) when compared to the control group with nonmodified mAb (CC49 1.34 ± 0.07% ID/mL). Conclusions: A diverse series of fast-reacting fluorinated d-TCOs were synthesized. A pretargeted blocking approach in tumor-bearing mice allowed the choice of a lead compound with fast reaction kinetics with Tz. A novel 18F-labeled d-TCO tracer was developed and used in a pretargeted PET imaging approach, allowing specific tumor visualization in a mouse model of colorectal cancer. Although further optimization of the radiotracer is needed to enhance the tumor-to-background ratios for pretargeted imaging, we anticipate that the 18F-labeled d-TCO will find use in studies where increased hydrophilicity and fast bioconjugation are required.
RESUMO
Background: To evaluate 18F-fluoro-2-deoxy-glucose (18F-FDG) and 18F-fluorothymidine (18F-FLT) as early-response biomarkers for phosphoinositide-3-kinase/Akt/mammalian-target-of-rapamycin (PI3K/Akt/mTOR) inhibition in breast cancer (BC) models. Materials and Methods: Two human epidermal growth factor receptor 2 (HER2)-positive (trastuzumab-sensitive SKBR3; trastuzumab-resistant JIMT1) and one triple-negative BC cell line (MDA-MB-231, trastuzumab, and everolimus resistant) were treated with trastuzumab (HER2 antagonist), PIK90 (PI3K inhibitor), or everolimus (mTOR inhibitor). Radiotracer uptake was measured before, 24, and 72 h after drug exposure and correlated with changes in cell number, glucose transporter 1 (GLUT1), cell cycle phase, and downstream signaling activation. Results: In responsive cells, cell number correlated with 18F-FLT at 24 h and 18F-FDG at 72 h of drug exposure, except in JIMT1 treated with everolimus, where both radiotracers failed to detect response owing to a temporary increase in tracer uptake. This flare can be caused by reflex activation of Akt combined with a hyperactive insulin-like growth factor I receptor (IGF-1R) signaling, resulting in increased trafficking of GLUTs to the cell membrane (18F-FDG) and enhanced DNA repair (18F-FLT). In resistant cells, no major changes were observed, although a nonsignificant flair for both tracers was observed in JIMT1 treated with trastuzumab. Conclusion: 18F-FLT positron emission tomography (PET) detects response to PI3K-targeting therapy earlier than 18F-FDG PET in BC cells. However, therapy response can be underestimated after trastuzumab and everolimus owing to negative feedback loop and crosstalk between pathways.
Assuntos
Neoplasias da Mama , Fluordesoxiglucose F18 , Animais , Humanos , Feminino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Everolimo/farmacologia , Everolimo/uso terapêutico , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/tratamento farmacológico , Serina-Treonina Quinases TOR , Trastuzumab , Tomografia por Emissão de Pósitrons/métodos , Linhagem Celular , Linhagem Celular Tumoral , Mamíferos/metabolismoRESUMO
BACKGROUND: CD70-CD27 is a costimulatory ligand-receptor pair in the tumor necrosis factor receptor family. With only limited expression in normal tissues, CD70 is constitutively expressed in a variety of solid tumors and hematologic malignancies, facilitating immunosuppression through CD27 signaling in the tumor microenvironment by enhanced survival of regulatory T cells, induction of T cell apoptosis, and T cell exhaustion. Consequently, CD70 is an increasingly recognized target for developing antibody-based therapies, but its expression patterns vary among different tumor types in spatial distribution, magnitude of expression and percentage of positive cells. In that regard, individual confirmation of CD70 expression at screening and during treatment could enhance the successful implementation of anti-CD70 therapies. Here, we developed a gallium-68 (68Ga) radiolabeled single-domain antibody-fragment targeting CD70 for in vivo positron emission tomography (PET) imaging. RESULTS: An anti-CD70 VHH construct containing a C-direct-tag with a free thiol was developed to enable site-specific conjugation to a NOTA bifunctional chelator for 68Ga radiolabeling. [68Ga]Ga-NOTA-anti-CD70 VHH was obtained in good radiochemical yield of 30.4 ± 1.7% and high radiochemical purity (> 94%). The radiolabeled VHH showed excellent in vitro and in vivo stability. Specific binding of [68Ga]Ga-NOTA-anti-CD70 VHH was observed on CD70high 786-O cells, showing significantly higher cell-associated activity when compared to the blocking condition (p < 0.0001) and CD70low NCl-H1975 cells (p < 0.0001). PET imaging showed specific radiotracer accumulation in CD70 expressing human tumor xenografts, which was efficiently blocked by prior injection of unlabeled anti-CD70 VHH (p = 0.0029). In addition, radiotracer uptake in CD70high tumors was significantly higher when compared with CD70low tumors (p < 0.0001). The distribution of the radioactivity in the tumors using autoradiography was spatially matched with immunohistochemistry analysis of CD70 expression. CONCLUSION: [68Ga]Ga-NOTA-anti-CD70 VHH showed excellent in vivo targeting of CD70 in human cancer xenografts. PET imaging using this radioimmunoconjugate holds promise as a non-invasive method to identify and longitudinally follow-up patients who will benefit most from anti-CD70 therapies.
RESUMO
BACKGROUND: Imaging of cell death can provide an early indication of treatment response in cancer. [99mTc]Tc-Duramycin is a small-peptide SPECT tracer that recognizes both apoptotic and necrotic cells by binding to phosphatidylethanolamine present in the cell membrane. Preclinically, this tracer has shown to have favorable pharmacokinetics and selective tumor accumulation early after the onset of anticancer therapy. In this first-in-human study, we report the safety, biodistribution and internal radiation dosimetry of [99mTc]Tc-Duramycin in healthy human volunteers. RESULTS: Six healthy volunteers (3 males, 3 females) were injected intravenously with [99mTc]Tc-Duramycin (dose: 6 MBq/kg; 473 ± 36 MBq). [99mTc]Tc-Duramycin was well tolerated in all subjects, with no serious adverse events reported. Following injection, a 30-min dynamic planar imaging of the abdomen was performed, and whole-body (WB) planar scans were acquired at 1, 2, 3, 6 and 23 h post-injection (PI), with SPECT acquisitions after each WB scan and one low-dose CT after the first SPECT. In vivo 99mTc activities were determined from semi-quantitative analysis of the images, and time-activity curves were generated. Residence times were calculated from the dynamic and WB planar scans. The mean effective dose was 7.61 ± 0.75 µSv/MBq, with the kidneys receiving the highest absorbed dose (planar analysis: 43.82 ± 4.07 µGy/MBq, SPECT analysis: 19.72 ± 3.42 µGy/MBq), followed by liver and spleen. The median effective dose was 3.61 mSv (range, 2.85-4.14). The tracer cleared slowly from the blood (effective half-life of 2.0 ± 0.4 h) due to high plasma protein binding with < 5% free tracer 3 h PI. Excretion was almost exclusively renal. CONCLUSION: [99mTc]Tc-Duramycin demonstrated acceptable dosimetry (< 5 mSv) and a favorable safety profile. Due to slow blood clearance, optimal target-to-background ratios are expected 5 h PI. These data support the further assessment of [99mTc]Tc-Duramycin for clinical treatment response evaluation. TRIAL REGISTRATION: NCT05177640, Registered April 30, 2021, https://clinicaltrials.gov/study/NCT05177640 .
RESUMO
As apoptosis occurs over an interval of time after administration of apoptosis-inducing therapy in tumors, the changes in technetium 99m ((99m)Tc)-tricarbonyl (CO)3 His-annexin A5 (His-ann A5) accumulation over time were examined. Colo205-bearing mice were divided into six treatment groups: (1) control, (2) 5-fluorouracil (5-FU; 250 mg/kg), (3) irinotecan (100 mg/kg), (4) oxaliplatin (30 mg/kg), (5) bevacizumab (5 mg/kg), and (6) panitumumab (6 mg/kg). (99m)Tc-(CO)3 His-ann A5 was injected 4, 8, 12, 24, and 48 hours posttreatment, and micro-single-photon emission computed tomography was performed. Immunostaining of caspase-3 (apoptosis), survivin (antiapoptosis), and LC3-II (autophagy marker) was also performed. Different dynamics of (99m)Tc-(CO)3 His-ann A5 uptake were observed in this colorectal cancer xenograft model, in response to a single dose of three different chemotherapeutics (5-FU, irinotecan, and oxaliplatin). Bevacizumab-treated mice showed no increased uptake of the radiotracer, and a peak of (99m)Tc-(CO)3 His-ann A5 uptake in panitumumab-treated mice was observed 24 hours posttreatment, as confirmed by caspase-3 immunostaining. For irinotecan-, oxaliplatin-, and bevacizumab-treated tumors, a significant correlation was established between the radiotracer uptake and caspase-3 immunostaining (r â=â .8, p < .05; r â=â .9, p < .001; r â=â .9, p < .001, respectively). For 5-FU- and panitumumab-treated mice, the correlation coefficients were r â=â .7 (p â=â .18) and r â=â .7 (p â=â .19), respectively. Optimal timing of annexin A5 imaging after the start of different treatments in the Colo205 model was determined.
Assuntos
Anexina A5 , Antineoplásicos/farmacologia , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/patologia , Compostos de Organotecnécio , Tecnécio , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos/uso terapêutico , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Humanos , Imuno-Histoquímica , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Repressoras/metabolismo , Survivina , Fatores de Tempo , Carga Tumoral/efeitos dos fármacosRESUMO
PURPOSE: RANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [89Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [64Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL. EXPERIMENTAL DESIGN: Fab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with 64Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated tumor targeting and biodistribution in transduced RANKL-ME-180 xenografts. RESULTS: The radiolabeling yield of [64Cu]Cu-NOTA-denos-Fab was 58 ± 9.2%, with a specific activity of 0.79 ± 0.11 MBq/µg (n = 3). A radiotracer binding assay proved specific targeting of RANKL in vitro. PET imaging showed fast blood clearance and high tumor accumulation as early as 1 h p.i. (2.14 ± 0.21% ID/mL), which peaked at 5 h p.i. (2.72 ± 0.61% ID/mL). In contrast, [64Cu]Cu-NOTA-denosumab reached its highest tumor uptake at 24 h p.i. (6.88 ± 1.12% ID/mL). [64Cu]Cu-NOTA-denos-Fab specifically targeted human RANKL in transduced ME-180 xenografts compared with the blocking group and negative ME-180 xenograft model. Histological analysis confirmed RANKL expression in RANKL-ME-180 xenografts. CONCLUSIONS: Here, we report on a novel RANKL PET imaging agent, [64Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications.
RESUMO
In this review, data on noninvasive imaging of apoptosis in oncology are reviewed. Imaging data available are presented in order of occurrence in time of enzymatic and morphologic events occurring during apoptosis. Available studies suggest that various radiopharmaceutical probes bear great potential for apoptosis imaging by means of positron emission tomography and single-photon emission computed tomography (SPECT). However, for several of these probes, thorough toxicologic studies are required before they can be applied in clinical studies. Both preclinical and clinical studies support the notion that 99mTc-hydrazinonicotinamide-annexin A5 and SPECT allow for noninvasive, repetitive, quantitative apoptosis imaging and for assessing tumor response as early as 24 hours following treatment instigation. Bioluminescence imaging and near-infrared fluorescence imaging have shown great potential in small-animal imaging, but their usefulness for in vivo imaging in humans is limited to structures superficially located in the human body. Although preclinical tumor-based data using high-frequency-ultrasonography (US) are promising, whether or not US will become a routinely clinically useful tool in the assessment of therapy response in oncology remains to be proven. The potential of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) for imaging late apoptotic processes is currently unclear. Neither 31P MRS nor 1H MRS signals seems to be a unique identifier for apoptosis. Although MRI-measured apparent diffusion coefficients are altered in response to therapies that induce apoptosis, they are also altered by nonapoptotic cell death, including necrosis and mitotic catastrophe. In the future, rapid progress in the field of apoptosis imaging in oncology is expected.
Assuntos
Apoptose/fisiologia , Diagnóstico por Imagem , Neoplasias/patologia , Animais , Feminino , Humanos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Neoplasias/química , Tomografia Computadorizada de EmissãoRESUMO
PURPOSE: The involvement of RANK/RANKL signaling in the tumor microenvironment (TME) in driving response or resistance to immunotherapy has only very recently been recognized. Current quantification methods of RANKL expression suffer from issues such as sensitivity, variability, and uncertainty on the spatial heterogeneity within the TME, resulting in conflicting reports on its reliability and limited use in clinical practice. Non-invasive molecular imaging using immuno-PET is a promising approach combining superior targeting specificity of monoclonal antibodies (mAb) and spatial, temporal and functional information of PET. Here, we evaluated radiolabeled anti-RANKL mAbs as a non-invasive biomarker of RANKL expression in the TME. EXPERIMENTAL DESIGN: Anti-human RANKL mAbs (AMG161 and AMG162) were radiolabeled with 89Zr using the bifunctional chelator DFO in high yield, purity and with intact binding affinity. After assessing the biodistribution in healthy CD-1 nude mice, [89Zr]Zr-DFO-AMG162 was selected for further evaluation in ME-180 (RANKL-transduced), UM-SCC-22B (RANKL-positive) and HCT-116 (RANKL-negative) human cancer xenografts to assess the feasibility of in vivo immuno-PET imaging of RANKL. RESULTS: [89Zr]Zr-DFO-AMG162 was selected as the most promising tracer for further validation based on biodistribution experiments. We demonstrated specific accumulation of [89Zr]Zr-DFO-AMG162 in RANKL transduced ME-180 xenografts. In UM-SCC-22B xenograft models expressing physiological RANKL levels, [89Zr]Zr-DFO-AMG162 imaging detected significantly higher signal compared to control [89Zr]Zr-DFO-IgG2 and to RANKL negative HCT-116 xenografts. There was good visual agreement with tumor autoradiography and immunohistochemistry on adjacent slides, confirming these findings. CONCLUSIONS: [89Zr]Zr-DFO-AMG162 can detect heterogeneous RANKL expression in the TME of human cancer xenografts, supporting further translation of RANKL immuno-PET to evaluate tumor RANKL distribution in patients.
RESUMO
Resistance against anti-cancer therapy is one of the major challenges during treatment of multiple cancers. Gemcitabine is a standard first-line chemotherapeutic drug, yet autophagy is highly activated in the hypoxic microenvironment of solid tumors and enhances the survival of tumor cells against gemcitabine chemotherapy. Recently, we showed the add-on effect of autophagy inhibitor UAMC-2526 to prevent HT-29 colorectal tumor growth in CD1-/- Foxn1nu mice treated with oxaliplatin. In this study, we aimed to investigate the potential beneficial effects of UAMC-2526 in a syngeneic Panc02 mouse model of pancreatic ductal adenocarcinoma (PDAC). Our data showed that UAMC-2526 combined with gemcitabine significantly reduced tumor growth as compared to the individual treatments. However, in contrast to in vitro experiments with Panc02 cells in culture, we were unable to detect autophagy inhibition by UAMC-2526 in Panc02 tumor tissue, neither via western blot analysis of autophagy markers LC3 and p62, nor by transmission electron microscopy. In vitro experiments revealed that UAMC-2526 enhances the potential of gemcitabine to inhibit Panc02 cell proliferation without obvious induction of cell death. Altogether, we conclude that although the combination treatment of UAMC-2526 with gemcitabine did not inhibit autophagy in the Panc02 mouse model, it has a beneficial effect on tumor growth inhibition.
RESUMO
The Ly49 natural killer (NK)-cell receptor family comprises both activating and inhibitory members, which recognize major histocompatibility complex (MHC) class I or MHC class I-related molecules and are involved in target recognition. As previously shown, the Ly49E receptor fails to bind to a variety of soluble or cell-bound MHC class I molecules, indicating that its ligand is not an MHC class I molecule. Using BWZ.36 reporter cells, we demonstrate triggering of Ly49E by the completely distinct, non-MHC-related protein urokinase plasminogen activator (uPA). uPA is known to be secreted by a variety of cells, including epithelial and hematopoietic cells, and levels are up-regulated during tissue remodeling, infections, and tumorigenesis. Here we show that addition of uPA to Ly49E-positive adult and fetal NK cells inhibits interferon-gamma secretion and reduces their cytotoxic potential, respectively. These uPA-mediated effects are Ly49E-dependent, as they are reversed by addition of anti-Ly49E monoclonal antibody and by down-regulation of Ly49E expression using RNA interference. Our results suggest that uPA, besides its established role in fibrinolysis, tissue remodeling, and tumor metastasis, could be involved in NK cell-mediated immune surveillance and tumor escape.
Assuntos
Células Matadoras Naturais/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Animais , Citotoxicidade Imunológica , Vigilância Imunológica , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Evasão TumoralRESUMO
Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are molecular imaging strategies that typically use radioactively labeled ligands to selectively visualize molecular targets. The nanomolar sensitivity of PET and SPECT combined with the high specificity and affinity of monoclonal antibodies have shown great potential in oncology imaging. Over the past decades a wide range of radio-isotopes have been developed into immuno-SPECT/PET imaging agents, made possible by novel conjugation strategies (e.g., site-specific labeling, click chemistry) and optimization and development of novel radiochemistry procedures. In addition, new strategies such as pretargeting and the use of antibody fragments have entered the field of immuno-PET/SPECT expanding the range of imaging applications. Non-invasive imaging techniques revealing tumor antigen biodistribution, expression and heterogeneity have the potential to contribute to disease diagnosis, therapy selection, patient stratification and therapy response prediction achieving personalized treatments for each patient and therefore assisting in clinical decision making.
RESUMO
Pretargeted positron emission tomography (PET) imaging based on the bioorthogonal inverse-electron-demand Diels-Alder reaction between tetrazines (Tz) and trans-cyclooctenes (TCO) has emerged as a promising tool for solid tumor imaging, allowing the use of short-lived radionuclides in immune-PET applications. With this strategy, it became possible to achieve desirable target-to-background ratios and at the same time to decrease the radiation burden to nontargeted tissues because of the fast clearance of small PET probes. Here, we show the synthesis of novel 18F-labeled dTCO-amide probes for pretargeted immuno-PET imaging. The PET probes were evaluated regarding their stability, reactivity toward tetrazine, and pharmacokinetic profile. [ 18 F]MICA-213 showed an extremely fast kinetic rate (10,553 M-1 s-1 in 50:50 MeOH/water), good stability in saline and plasma up to 4 h at 37 °C with no isomerization observed, and the biodistribution in healthy mice revealed a mixed hepatobiliary and renal clearance with no defluorination and low background in other tissues. [ 18 F]MICA-213 was further used for in vivo pretargeted immune-PET imaging carried out in nude mice bearing LS174T colorectal tumors that were previously treated with a tetrazine-modified anti-TAG-72 monoclonal antibody (CC49). Pretargeted µPET imaging results showed clear visualization of the tumor tissue with a significantly higher uptake when compared to the control.
RESUMO
BACKGROUND: Fibroblast activation protein (FAP) is a proline selective serine protease that is overexpressed in tumor stroma and in lesions of many other diseases that are characterized by tissue remodeling. In 2014, a most potent FAP-inhibitor (referred to as UAMC1110) with low nanomolar FAP-affinity and high selectivity toward related enzymes such as prolyl oligopeptidase (PREP) and the dipeptidyl-peptidases (DPPs): DPP4, DPP8/9 and DPP2 were developed. This inhibitor has been adopted recently by other groups to create radiopharmaceuticals by coupling bifunctional chelator-linker systems. Here, we report squaric acid (SA) containing bifunctional DATA5m and DOTA chelators based on UAMC1110 as pharmacophor. The novel radiopharmaceuticals DOTA.SA.FAPi and DATA5m.SA.FAPi with their non-radioactive derivatives were characterized for in vitro inhibitory efficiency to FAP and PREP, respectively and radiochemical investigated with gallium-68. Further, first proof-of-concept in vivo animal study followed by ex vivo biodistribution were determined with [68Ga]Ga-DOTA.SA.FAPi. RESULTS: [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DATA5m.SA.FAPi showed high complexation > 97% radiochemical yields after already 10 min and high stability over a period of 2 h. Affinity to FAP of DOTA.SA.FAPi and DATA5m.SA.FAPi and its natGa and natLu-labeled derivatives were excellent resulting in low nanomolar IC50 values of 0.7-1.4 nM. Additionally, all five compounds showed low affinity for the related protease PREP (high IC50 with 1.7-8.7 µM). First proof-of-principle in vivo PET-imaging animal studies of the [68Ga]Ga-DOTA.SA.FAPi precursor in a HT-29 human colorectal cancer xenograft mouse model indicated promising results with high accumulation in tumor (SUVmean of 0.75) and low background signal. Ex vivo biodistribution showed highest uptake in tumor (5.2%ID/g) at 60 min post injection with overall low uptake in healthy tissues. CONCLUSION: In this work, novel PET radiotracers targeting fibroblast activation protein were synthesized and biochemically investigated. Critical substructures of the novel compounds are a squaramide linker unit derived from the basic motif of squaric acid, DOTA and DATA5m bifunctional chelators and a FAP-targeting moiety. In conclusion, these new FAP-ligands appear promising, both for further research and development as well as for first human application.