RESUMEN
Very late antigen-4 (VLA4; CD49d) is a promising immune therapy target in treatment-resistant leukemia and multiple myeloma, and there is growing interest in repurposing the humanized monoclonal antibody (Ab), natalizumab, for this purpose. Positron emission tomography with radiolabeled Abs (immuno-PET) could facilitate this effort by providing information on natalizumab's in vivo pharmacokinetic and target delivery properties. In this study, we labeled natalizumab with 89Zr specifically on sulfhydryl moieties via maleimide-deferoxamine conjugation. High VLA4-expressing MOLT4 human T cell acute lymphoblastic leukemia cells showed specific 89Zr-natalizumab binding that was markedly blocked by excess Ab. In nude mice bearing MOLT4 tumors, 89Zr-natalizumab PET showed high-contrast tumor uptake at 7 days postinjection. Biodistribution studies confirmed that uptake was the highest in MOLT4 tumors (2.22 ± 0.41%ID/g) and the liver (2.33 ± 0.76%ID/g), followed by the spleen (1.51 ± 0.42%ID/g), while blood activity was lower at 1.12 ± 0.21%ID/g. VLA4-specific targeting in vivo was confirmed by a 58.1% suppression of tumor uptake (0.93 ± 0.15%ID/g) when excess Ab was injected 1 h earlier. In cultured MOLT4 cells, short-term 3 day exposure to the proteasome inhibitor bortezomib (BTZ) did not affect the α4 integrin level, but BTZ-resistant cells that survived the treatment showed increased α4 integrin expression. When the effects of BTZ treatment were tested in mice, there was no change of the α4 integrin level or 89Zr-natalizumab uptake in MOLT4 leukemia tumors, which underscores the complexity of tumor VLA4 regulation in vivo. In conclusion, 89Zr-natalizumab PET may be useful for noninvasive monitoring of tumor VLA4 and may assist in a more rational application of Ab-based therapies for hematologic malignancies.
Asunto(s)
Integrina alfa4beta1 , Leucemia , Humanos , Animales , Ratones , Natalizumab/uso terapéutico , Cisteína , Integrina alfa4 , Ratones Desnudos , Distribución Tisular , Línea Celular Tumoral , Tomografía de Emisión de Positrones/métodos , Circonio/químicaRESUMEN
We developed an immuno-PET technique that monitors modulation of tumor CD133 expression, which is required for the success of CD133-targeted therapies. Methods. Anti-CD133 antibodies were subjected to sulfhydryl moiety-specific 89Zr conjugation. 89Zr-CD133 IgG was evaluated for specific activity and radiolabel stability. Colon cancer cells underwent binding assays and Western blotting. Biodistribution and PET studies were performed in mice. Results. 89Zr-CD133 IgG showed excellent target specificity with 97.2 ± 0.7% blocking of HT29 cell binding by an excess antibody. Intravenous 89Zr-CD133 IgG followed biexponential blood clearance and showed CD133-specific uptake in HT29 tumors. 89Zr-CD133 IgG PET/CT and biodistribution studies confirmed high HT29 tumor uptake with lower activities in the blood and normal organs. In HT29 cells, celecoxib dose-dependently decreased CD133 expression and 89Zr-CD133 IgG binding that reached 19.9 ± 2.1% (P < 0.005) and 50.3 ± 10.9% (P < 0.001) of baseline levels by 50 µM, respectively. Celecoxib treatment of mice significantly suppressed tumor CD133 expression to 67.5 ± 7.8% of controls (P < 0.005) and reduced tumor 89Zr-CD133 IgG uptake from 15.5 ± 1.4% at baseline to 12.3 ± 2.0%ID/g (P < 0.01). Celecoxib-induced CD133 reduction in HT29 cells and tumors was associated with substantial suppression of AKT activation. There were also reduced HIF-1α accumulation and IκBα/NFκB phosphorylation. Conclusion. 89Zr-CD133 IgG PET provides high-contrast tumor imaging and monitors celecoxib treatment-induced modulation of tumor CD133 expression, which was found to occur through AKT inhibition. This technique may thus be useful for screening drugs that can effectively suppress colon cancer stem cells.
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Neoplasias del Colon , Animales , Celecoxib/farmacología , Línea Celular Tumoral , Neoplasias del Colon/diagnóstico por imagen , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Humanos , Inmunoglobulina G , Ratones , Células Madre Neoplásicas , Tomografía Computarizada por Tomografía de Emisión de Positrones , Tomografía de Emisión de Positrones/métodos , Proteínas Proto-Oncogénicas c-akt , Distribución Tisular , CirconioRESUMEN
Immune therapy of T-cell lymphoma requires assessment of tumor-expressed programmed cell death protein-1 (PD-1). Herein, we developed an immuno-PET technique that quantitatively images and monitors regulation of PD-1 expression on T-cell lymphomas. Methods. Anti-PD-1 IgG underwent sulfhydryl moiety-specific conjugation with maleimide-deferoxamine and 89Zr labeling. Binding assays and Western blotting were performed in EL4 murine T-cell lymphoma cells. In vivo pharmacokinetics, biodistribution, and PET were performed in mice. Results. 89Zr-PD-1 IgG binding to EL4 cells was completely blocked by cold antibodies, confirming excellent target specificity. Following intravenous injection into mice, 89Zr-PD-1 IgG showed biexponential blood clearance and relatively low normal organ uptake after five days. PET/CT and biodistribution demonstrated high EL4 tumor uptake that was suppressed by cold antibodies. In EL4 cells, phorbol 12-myristate 13-acetate (PMA) increased 89Zr-PD-1 IgG binding (305.5 ± 30.6%) and dose-dependent augmentation of PD-1 expression (15.8 ± 3.8 - fold of controls by 200 ng/ml). FACS showed strong PD-1 expression on all EL4 cells and positive but weaker expression on 41.6 ± 2.1% of the mouse spleen lymphocytes. PMA stimulation led to 2.7 ± 0.3-fold increase in the proportion of the strongest PD-1 expressing EL4 cells but failed to influence that of PD-1+ mouse lymphocytes. In mice, PMA treatment increased 89Zr-PD-1 IgG uptake in EL4 lymphomas from 6.6 ± 1.6 to 13.9 ± 3.6%ID/g (P = 0.01), and tumor uptake closely correlated with PD-1 level (r = 0.771, P < 0.001). On immunohistochemistry of tumor sections, infiltrating CD8α+ T lymphocytes constituted a small fraction of tumor cells. The entire tumor section showed strong PD-1 staining that was even stronger for PMA-treated mice. Investigation of involved signaling revealed that PMA increased EL4 cell and tumor HIF-1α accumulation and NFκB and JNK activation. Conclusion. 89Zr-PD-1 IgG offered high-contrast PET imaging of tumor PD-1 in mice. This was found to mostly represent binding to EL4 tumor cells, although infiltrating T lymphocytes may also have contributed. PD-1 expression on T-cell lymphomas was upregulated by PMA stimulation, and this was reliably monitored by 89Zr-PD-1 IgG PET. This technique may thus be useful for understanding the mechanisms of PD-1 regulation in lymphomas of living subjects.
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Linfoma de Células T , Linfoma , Animales , Línea Celular Tumoral , Humanos , Inmunoglobulina G/metabolismo , Linfoma/diagnóstico por imagen , Linfoma/metabolismo , Linfoma/patología , Ratones , Tomografía Computarizada por Tomografía de Emisión de Positrones , Tomografía de Emisión de Positrones/métodos , Receptor de Muerte Celular Programada 1/metabolismo , Acetato de Tetradecanoilforbol , Distribución Tisular , CirconioRESUMEN
Monoclonal antibodies (Ab) have revolutionized the management of lymphomas, the most common hematologic malignancy in adults. Indeed, incorporation of rituximab into the regimen for indolent non-Hodgkin's lymphomas (NHLs) has dramatically improved treatment response and disease outcome. Yet, newer Ab therapeutics against promising antigen targets need to be developed to treat refractory or relapsed patients. Treatment efficacy can be further enhanced by conjugating toxic molecules to the Abs. Radioimmunotherapy (RIT) harnesses Abs as vehicles for targeted delivery of therapeutic radionuclide payloads for direct killing of targeted tumor cells. Positron emission tomography (PET) with radiolabeled Abs (called immuno-PET) can facilitate the development of new Ab therapeutics and RIT by providing pharmacokinetic and pharmacodynamic information and by quantifying tumor antigen level relevant for treatment decision. Immuno-PET has recently gravitated toward labeling Abs with 89Zr, a radiometal with a 3.3 day half-life that is trapped following Ab internalization and thus provides high-resolution PET images with excellent contrast. Immuno-PET methods against major lymphoma antigens including CD20 and other promising targets are currently under development. With continued improvements, immuno-PET has the potential to be used in lymphoma management as an imaging biomarker for patient selection and assessment of treatment response.
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Linfoma , Radioinmunoterapia , Adulto , Anticuerpos Monoclonales , Antígenos de Neoplasias , Humanos , Linfoma/diagnóstico por imagen , Linfoma/tratamiento farmacológico , Linfoma/radioterapia , Tomografía de Emisión de Positrones , Radioinmunoterapia/métodos , Radioisótopos/uso terapéutico , RituximabRESUMEN
Acute ß-adrenergic stimulation contributes to heart failure. Here, we investigated the role of p53 in isoproterenol (ISO)-mediated metabolic and oxidative stress effects on cardiomyocytes and explored the direct protective effects offered by the antioxidant nutraceutical curcumin. Differentiated H9C2 rat cardiomyocytes treated with ISO were assayed for glucose uptake, lactate release, and mitochondrial reactive oxygen species (ROS) generation. Survival was assessed by sulforhodamine B assays. Cardiomyocytes showed significantly decreased glucose uptake and lactate release, as well as increased cellular toxicity by ISO treatment. This was accompanied by marked dose-dependent increases of mitochondria-derived ROS. Scavenging with N-acetyl-L-cysteine (NAC) effectively lowered ROS levels, which completely recovered glycolytic metabolism and survival suppressed by ISO. Mechanistically, ISO reduced extracellular-signal-regulated kinase (ERK) activation, whereas it upregulated p53 expression in an ROS-dependent manner. Silencing of p53 with siRNA blocked the ability of ISO to stimulate mitochondrial ROS and suppress glucose uptake, and partially recovered cell survival. Finally, curcumin completely reversed the metabolic and ROS-stimulating effects of ISO. Furthermore, curcumin improved survival of cardiomyocytes exposed to ISO. Thus, ISO suppresses cardiomyocyte glycolytic metabolism and survival by stimulating mitochondrial ROS in a p53-dependent manner. Furthermore, curcumin can efficiently rescue cardiomyocytes from these adverse effects.
Asunto(s)
Curcumina/farmacología , Isoproterenol/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Antioxidantes/farmacología , Cardiotónicos/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucosa/metabolismo , Isoproterenol/efectos adversos , Estrés Oxidativo/efectos de los fármacos , RatasRESUMEN
The role of apoptosis imaging for monitoring treatment response in ischemic limbs has not been properly explored. In this study, we investigated the ability of annexin V (AnxV) imaging to assess the efficacy of antiapoptotic treatment in ischemic limbs of diabetic mice. Normal C57BL/6 mice and streptozotocin-induced diabetic mice were subject to hindlimb ischemia. AnxV-conjugated fluorescent streptavidin probes were intravenously injected, and optical imaging was performed. Tissue apoptosis was quantified by histochemistry and Western blotting. The AnxV probes showed specific targeting to apoptotic cells on confocal microscopy and flow cytometry. Intravenous AnxV probes displayed substantially greater accumulation in ischemic limbs of diabetic mice. Benfotiamine (BFT) treatment of diabetic mice led to better perfusion recovery on laser Doppler imaging and reduced AnxV binding on optical imaging. TUNEL staining and cleaved caspase-3 Western blots confirmed accelerated apoptosis by diabetes and its suppression by BFT treatment. Furthermore, AnxV-SAv-PEcy5.5 uptake in the ischemic limbs closely correlated to cleaved caspase-3 expression. Thus, AnxV imaging may be useful for monitoring the efficacy of therapeutic agents designed to suppress ischemia-induced apoptosis.
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Anexina A5/farmacología , Miembro Posterior/patología , Isquemia/tratamiento farmacológico , Isquemia/patología , Tiamina/análogos & derivados , Animales , Anexina A5/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Miembro Posterior/irrigación sanguínea , Flujometría por Láser-Doppler , Ratones , Ratones Endogámicos C57BL , Estreptavidina/administración & dosificación , Estreptavidina/farmacología , Tiamina/administración & dosificación , Tiamina/uso terapéuticoRESUMEN
CD73 is a cell-surface ectoenzyme that hydrolyzes the conversion of extracellular adenosine monophosphate to adenosine, which in turn can promote resistance to immune checkpoint blockade therapy. Immune response may therefore be improved by targeting tumor CD73, and this possibility underlines the need to non-invasively assess tumor CD73 level. In this study, we developed a cysteine site-specific 89Zr-labeled anti-CD73 (89Zr-CD73) IgG immuno-PET technique that can image tumor CD73 expression in living bodies. Anti-CD73 IgG was reduced with tris(2-carboxyethyl)phosphine, underwent sulfohydryl moiety-specific conjugation with deferoxamine-maleimide, and was radiolabeled with 89Zr. CT26 mouse colon cancer cells, CT26/CD73 cells engineered to constitutively overexpress CD73, and 4T1.2 mouse breast cancer cells underwent cell binding assays and western blotting. Balb/c nude mice bearing tumors underwent 89Zr-CD73 IgG PET imaging and biodistribution studies. 89Zr-CD73 IgG showed 20-fold higher binding to overexpressing CT26/CD73 cells compared to low-expressing CT26 cells, and moderate expressing 4T1.2 cells showed uptake that was 38.9 ± 1.51% of CT26/CD73 cells. Uptake was dramatically suppressed by excess unlabeled antibody. CD73 content proportionately increased in CT26 and CT26/CD73 cell mixtures was associated with linear increases in 89Zr-CD73 IgG uptake. 89Zr-CD73 IgG PET/CT displayed clear accumulation in CT26/CD73 tumors with greater uptake compared to CT26 tumors (3.13 ± 1.70%ID/g vs. 1.27 ± 0.31%ID/g at 8 days; P = 0.04). Specificity was further supported by low CT26/CD73 tumor-to-blood ratio of 89Zr-isotype-IgG compared to 89Zr-CD73 IgG (0.48 ± 0.08 vs. 2.68 ± 0.52 at 4 days and 0.53 ± 0.07 vs. 4.81 ± 1.02 at 8 days; both P < 0.001). Immunoblotting and immunohistochemistry confirmed strong CD73 expression in CT26/CD73 tumors and low expression in CT26 tumors. 4T1.2 tumor mice also showed clear 89Zr-CD73 IgG accumulation at 8 days (3.75 ± 0.70%ID/g) with high tumor-to-blood ratio compared to 89Zr-isotype-IgG (4.91 ± 1.74 vs. 1.20 ± 0.28; P < 0.005). 89Zr-CD73 IgG specifically targeted CD73 on high expressing cancer cells in vitro and tumors in vivo. Thus, 89Zr-CD73 IgG immuno-PET may be useful for the non-invasive monitoring of CD73 expression in tumors of living subjects.
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5'-Nucleotidasa , Neoplasias del Colon , Cisteína , Tomografía de Emisión de Positrones , Circonio , Animales , 5'-Nucleotidasa/metabolismo , Circonio/química , Neoplasias del Colon/diagnóstico por imagen , Neoplasias del Colon/metabolismo , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Ratones , Línea Celular Tumoral , Tomografía de Emisión de Positrones/métodos , Cisteína/metabolismo , Humanos , Radioisótopos , Femenino , Ratones Endogámicos BALB C , Distribución Tisular , Ratones Desnudos , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismoRESUMEN
An efficient and straightforward method for radiolabeling nanoparticles is urgently needed to understand the in vivo biodistribution of nanoparticles. Herein, we investigated a facile and highly efficient strategy to prepare radiolabeled glycol chitosan nanoparticles with (64)Cu via a strain-promoted azide-alkyne cycloaddition strategy, which is often referred to as click chemistry. First, the azide (N3) group, which allows for the preparation of radiolabeled nanoparticles by copper-free click chemistry, was incorporated to glycol chitosan nanoparticles (CNPs). Second, the strained cyclooctyne derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator, was synthesized for preparing the preradiolabeled alkyne complex with (64)Cu radionuclide. Following incubation with the (64)Cu-radiolabeled DBCO complex (DBCO-PEG4-Lys-DOTA-(64)Cu with high specific activity, 18.5 GBq/µmol), the azide-functionalized CNPs were radiolabeled successfully with (64)Cu, with a high radiolabeling efficiency and a high radiolabeling yield (>98%). Importantly, the radiolabeling of CNPs by copper-free click chemistry was accomplished within 30 min, with great efficiency in aqueous conditions. In addition, we found that the (64)Cu-radiolabeled CNPs ((64)Cu-CNPs) did not show any significant effect on the physicochemical properties, such as size, zeta potential, or spherical morphology. After (64)Cu-CNPs were intravenously administered to tumor-bearing mice, the real-time, in vivo biodistribution and tumor-targeting ability of (64)Cu-CNPs were quantitatively evaluated by microPET images of tumor-bearing mice. These results demonstrate the benefit of copper-free click chemistry as a facile, preradiolabeling approach to conveniently radiolabel nanoparticles for evaluating the real-time in vivo biodistribution of nanoparticles.
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Quitosano/química , Química Clic/métodos , Radioisótopos de Cobre/química , Nanopartículas/química , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones DesnudosRESUMEN
We aimed comparing two-year clinical outcomes of the Everolimus-Eluting Promus and Paclitaxel-Eluting TAXUS Liberte stents used in routine clinical practice. Patients with objective evidence of ischemia and coronary artery disease eligible for PCI were prospectively randomized to everolimus-eluting stent (EES) or paclitaxel-eluting stent (PES) groups. The primary end-point was ischemia-driven target vessel revascularization (TVR) at 2 yr after intervention, and the secondary end-point was a major adverse cardiac event (MACE), such as death, myocardial infarction (MI), target lesion revascularization (TLR), TVR or stent thrombosis. A total of 850 patients with 1,039 lesions was randomized to the EES (n=425) and PES (n=425) groups. Ischemic-driven TVR at 2 yr was 3.8% in the PES and 1.2% in the EES group (P for non-inferiority=0.021). MACE rates were significantly different; 5.6% in PES and 2.5% in EES (P = 0.027). Rates of MI (0.8% in PES vs 0.2% in EES, P = 0.308), all deaths (1.5% in PES vs 1.2% in EES, P = 0.739) and stent thrombosis (0.3% in PES vs 0.7% in EES, P = 0.325) were similar. The clinical outcomes of EES are superior to PES, mainly due to a reduction in the rate of ischemia-driven TVR.
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Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Stents Liberadores de Fármacos , Paclitaxel/uso terapéutico , Intervención Coronaria Percutánea/métodos , Sirolimus/análogos & derivados , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/uso terapéutico , Enfermedad de la Arteria Coronaria/mortalidad , Reestenosis Coronaria/prevención & control , Everolimus , Femenino , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/uso terapéutico , Masculino , Persona de Mediana Edad , Paclitaxel/administración & dosificación , Estudios Prospectivos , Sirolimus/administración & dosificación , Sirolimus/uso terapéutico , Trombosis , Resultado del TratamientoRESUMEN
In this study, we investigated the effects of hydrazinonicotinamide (HYNIC)-a bifunctional crosslinker widely used to (99m)Tc radiolabel protein and nanoparticles for imaging studies-on quantum dot opsonization, macrophage engulfment and in vivo kinetics. In streptavidin-coated quantum dots (SA-QDots), conjugation with HYNIC increased the net negative charge without affecting the zeta potential. Confocal microscopy and fluorescence-activated cell sorting showed HYNIC attachment to suppress SA-QDot engulfment by macrophages. Furthermore, HYNIC conjugation suppressed surface opsonization by serum protein including IgG. When intravenously injected into mice, HYNIC conjugation significantly prolonged the circulation of SA-QDots and reduced their hepatosplenic uptake. Diminished reticuloendothelial system clearance of SA-QDots and aminoPEG-QDots by HYNIC conjugation was also demonstrated by in vivo and ex vivo optical imaging. The effects of HYNIC on the opsonization, phagocytosis and in vivo kinetics of quantum dots were reversed by removal of the hydrazine component from HYNIC. Thus, surface functionalization with HYNIC can improve the in vivo kinetics of quantum dots by reducing phagocytosis via suppression of surface opsonization.
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Sistema Mononuclear Fagocítico/fisiología , Niacinamida/análogos & derivados , Proteínas Opsoninas/metabolismo , Fagocitosis/fisiología , Puntos Cuánticos , Animales , Células Cultivadas , Reactivos de Enlaces Cruzados/química , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Niacinamida/químicaRESUMEN
Introduction: Positron emission tomography (PET) using radiolabeled Abs as imaging tracer is called immuno-PET. Immuno-PET can verify therapeutic Ab delivery and can noninvasively quantify global levels of target expression in tumors of living subjects. The interleukin-2 receptor α chain (IL-2Rα; CD25) is a promising target for immune therapy and radioimmunotherapy of lymphomas. Immuno-PET could facilitate this approach by visualizing CD25 expression in vivo. Methods: We prepared 89Zr-anti-CD25 IgG specifically labeled to sulfhydryl moieties by maleimide-deferoxamine conjugation. Results and Discussion: CD25(+) SUDHL1 human T-cell lymphoma cells showed high anti-human 89Zr-CD25 IgG binding that reached 32-fold of that of CD25(-) human lymphoma cells and was completely blocked by excess unlabeled Ab. In SUDHL1 tumor-bearing nude mice, pharmacokinetic studies demonstrated exponential reductions of whole blood and plasma activity following intravenous 89Zr-anti-CD25 IgG injection, with half-lives of 26.0 and 23.3 h, respectively. SUDHL1 tumor uptake of 89Zr-CD25 IgG was lower per weight in larger tumors, but blood activity did not correlate with tumor size or blood level of human CD25, indicating minimal influence by circulating soluble CD25 protein secreted from the lymphoma cells. 89Zr-CD25 IgG PET allowed high-contrast SUDHL1 lymphoma visualization at five days. Biodistribution studies confirmed high tumor 89Zr-CD25 IgG uptake (8.7 ± 0.9%ID/g) that was greater than blood (5.2 ± 1.6%ID/g) and organ uptakes (0.7 to 3.5%ID/g). Tumor CD25-specific targeting was confirmed by suppression of tumor uptake to 4.3 ± 0.2%ID by excess unlabeled CD25 IgG, as well as by low tumor uptake of 89Zr-labeled IgG2a isotype control Ab (3.6 ± 0.9%ID). Unlike CD25(+) lymphocytes from mouse thymus that showed specific uptake of anti-mouse 89Zr-CD25 IgG, EL4 mouse lymphoma cells had low CD25 expression and showed low uptake. In immunocompetent mice bearing EL4 tumors, anti-mouse 89Zr-CD25 IgG displayed low uptakes in normal organs as well as in the tumor. Furthermore, the biodistribution was not influenced by Ab blocking, indicating that specific uptake in nontumor tissues was minimal. 89Zr-CD25 IgG immuno-PET may thus be useful for imaging of T-cell lymphomas and noninvasive assessment of CD25 expression on target cells in vivo.
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Linfoma de Células T , Linfoma , Animales , Ratones , Humanos , Radioisótopos/farmacocinética , Subunidad alfa del Receptor de Interleucina-2 , Cisteína , Ratones Desnudos , Anticuerpos Monoclonales , Línea Celular Tumoral , Tomografía de Emisión de Positrones/métodos , Linfoma/patologíaRESUMEN
Extracellular vesicles (EVs) are a promising carrier for various cargos with antitumor effects, but their capacity to transfer the ability to transport radioiodine for cancer theranostics remains unexplored. Herein, we tested the hypothesis that EVs can be loaded with the sodium iodide symporter (NIS) protein and efficiently deliver the payload to recipient cancer cells to facilitate radioiodine uptake. The results revealed that donor cells either transduced with an adenoviral vector for transient expression or engineered for stable overexpression secreted EVs that contained substantial amounts of NIS protein but not NIS mRNA. Huh7 liver cancer cells treated with EVs secreted from each of the donor cell types showed significantly increased plasma membrane NIS protein, indicating efficient payload delivery. Furthermore, intact function of the delivered NIS protein was confirmed by significantly increased radioiodine transport in recipient cancer cells that peaked at 48 h. Importantly, NIS protein delivered by EVs significantly enhanced the antitumor effects of 131I radiotherapy. These results reveal that EVs are a promising vehicle to deliver NIS protein to cancer cells in sufficient amounts for radioiodine-based theranostics.
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Vesículas Extracelulares , Radioisótopos de Yodo , Neoplasias/radioterapia , Simportadores , Tratamiento Basado en Trasplante de Células y Tejidos , Excipientes , Humanos , Radioisótopos de Yodo/administración & dosificación , Proteínas de la Membrana , Neoplasias/metabolismo , Simportadores/administración & dosificación , Simportadores/metabolismoRESUMEN
OBJECTIVE: A better understanding of the metabolic phenotype of stem-like cancer cells could provide targets to help overcome chemoresistance. In this study, we hypothesized that colon cancer cells with the stem cell feature of CD133 expression have increased proton leakage that influences glucose metabolism and offers protection against reactive oxygen species (ROS)-inducing treatment. METHODS AND RESULTS: In HT29 colon cancer cells, 18 F-fluorodeoxyglucose (FDG) uptake was increased by CD133 selection and decreased by CD133 silencing. In CD133(+) cells, greater 18 F-FDG uptake was accompanied by increased oxygen consumption rate (OCR) and reduced mitochondrial membrane potential and mitochondrial ROS, indicating increased proton leakage. The uncoupling protein inhibitor genipin reversed the increased 18 F-FDG uptake and greater OCR of CD133(+) cells. The ROS-inducing drug, piperlongumine, suppressed CD133(-) cell survival by stimulating mitochondrial ROS generation but was unable to influence CD133(+) cells when used alone. However, cotreatment of CD133(+) cells with genipin and piperlongumine efficiently stimulated mitochondrial ROS for an enhanced antitumor effect with substantially reduced CD133 expression. CONCLUSION: These results demonstrate that mitochondrial uncoupling is a metabolic feature of CD133(+) colon cancer cells that provides protection against piperlongumine therapy by suppressing mitochondrial ROS generation. Hence, combining genipin with ROS-inducing treatment may be an effective strategy to reverse the metabolic feature and eliminate stem-like colon cancer cells.
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Neoplasias del Colon , Glucosa , Neoplasias del Colon/patología , Fluorodesoxiglucosa F18/metabolismo , Glucosa/metabolismo , Humanos , Estrés Oxidativo , Protones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
CD44 is a cell-surface glycoprotein involved in cell-cell interaction, adhesion, and migration. CD44 is found on colon cancer cells and on immune cells. Previous studies of 89Zr PET imaging of CD44 have relied on an anti-human antibody (Ab), which can influence biodistribution in murine models. In this study, we used an Ab that cross-reacts with both human and mouse origin CD44 of all isoforms to unveil the type of leukocyte responsible for high splenic anti-CD44 uptake and investigate how its regulation can influence tumor immuno-PET. The Ab was site-specifically labeled with 89Zr-deferoxamine on cysteine residues. 89Zr-anti-CD44 demonstrated high-specific binding to HT29 human colon cancer cells and monocytic cells that showed CD44 expression. When 89Zr-anti-CD44 was administered to Balb/C nude mice, there was remarkably high splenic uptake but low SNU-C5 tumor uptake (1.2 ± 0.7%ID/g). Among cells isolated from Balb/C mouse spleen, there was greater CD44 expression on CD11b positive myeloid cells than lymphocytes. In cultured monocytic and macrophage cells, LPS stimulation upregulated CD44 expression and increased 89Zr-anti-CD44 binding. Similarly, normal Balb/C mice that underwent lipopolysaccharide (LPS) stimulation showed a significant upregulation of CD44 expression on splenic myeloid cells. Furthermore, LPS treatment stimulated a 2.44-fold increase of 89Zr-anti-CD44 accumulation in the spleen, which was attributable to splenic myeloid cells. Finally, in Balb/C nude mice bearing HT29 tumors, we injected 89Zr-anti-CD44 with greater Ab doses to reduce binding to splenic cells. The results showed lower spleen uptake and improved tumor uptake (2.9 ± 1.3%ID/g) with a total of 300 µg of Ab dose, and further reduction of spleen uptake and greater tumor uptake (5.7 ± 0.0%ID/g) with 700 µg Ab dose. Thus, using an 89Zr labeled Ab that cross-reacts with both human and mouse CD44, we demonstrate that CD44 immuno-PET has the capacity to monitor CD44 regulation on splenic myeloid cells and may also be useful for imaging colon tumors.
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Receptores de Hialuranos/análisis , Leucocitos/metabolismo , Radioisótopos , Bazo/inmunología , Circonio , Animales , Anticuerpos , Deferoxamina , Células HT29 , Humanos , Receptores de Hialuranos/metabolismo , Ratones , Tomografía de Emisión de Positrones , Células RAW 264.7 , Bazo/metabolismoRESUMEN
We developed an 89Zr-labeled anti-programmed death ligand 1 (anti-PD-L1) immune PET that can monitor chemotherapy-mediated modulation of tumor PD-L1 expression in living subjects. Methods: Anti-PD-L1 underwent sulfohydryl moiety-specific conjugation with maleimide-deferoxamine followed by 89Zr radiolabeling. CT26 colon cancer cells and PD-L1-overexpressing CT26/PD-L1 cells underwent binding assays, flow cytometry, and Western blotting. In vivo pharmacokinetics, biodistribution, and PET imaging were evaluated in mice. Results:89Zr-anti-PD-L1 synthesis was straightforward and efficient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that reduction produced half-antibody fragments, and matrix-assisted laser desorption ionization time-of-flight analysis estimated 2.18 conjugations per antibody, indicating specific conjugation at the hinge-region disulfide bonds. CT26/PD-L1 cells showed 102.2 ± 6.7-fold greater 89Zr-anti-PD-L1 binding than that of weakly expressing CT26 cells. Excellent target specificity was confirmed by a drastic reduction in binding by excess cold antibody. Intravenous 89Zr-anti-PD-L1 followed biexponential blood clearance. PET/CT image analysis demonstrated decreases in major organ activity over 7 d, whereas high CT26/PD-L1 tumor activity was maintained. Again, this was suppressed by excess cold antibody. Treatment of CT26 cells with gemcitabine for 24 h augmented PD-L1 protein to 592.4% ± 114.2% of the control level and increased 89Zr-anti-PD-L1 binding, accompanied by increased AKT (protein kinase B) activation and reduced phosphatase and tensin homolog (PTEN). In CT26 tumor-bearing mice, gemcitabine treatment substantially increased tumor uptake from 1.56% ± 0.48% to 6.24% ± 0.37% injected dose per gram (tumor-to-blood ratio, 34.7). Immunoblots revealed significant increases in tumor PD-L1 and activated AKT and a decrease in PTEN. Conclusion:89Zr-anti-PD-L1 showed specific targeting with favorable imaging properties. Gemcitabine treatment upregulated cancer cell and tumor PD-L1 expression and increased 89Zr-anti-PD-L1 uptake. 89Zr-anti-PD-L1 PET may thus be useful for monitoring chemotherapy-mediated tumor PD-L1 modulation in living subjects.
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Antígeno B7-H1/metabolismo , Neoplasias del Colon/patología , Desoxicitidina/análogos & derivados , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inmunoconjugados/inmunología , Tomografía Computarizada por Tomografía de Emisión de Positrones , Radioisótopos , Circonio , Animales , Antígeno B7-H1/inmunología , Línea Celular Tumoral , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Inmunoconjugados/farmacocinética , Marcaje Isotópico , Ratones , Distribución Tisular , GemcitabinaRESUMEN
The uncoupling protein-2 (UCP2) serves a role in tumor aggressiveness and anticancer resistance, which is considered to be associated with its ability to attenuate reactive oxygen species (ROS) production. We hypothesized that UCP2 may protect cancer cells from elesclomol-induced cytotoxicity, and that this may be overcome by blocking UCP2 function with genipin. In A549 lung cancer cells that exhibited high UCP2 expression, treatment with elesclomol alone induced limited changes in glucose uptake, ROS production and cell survival. By contrast, both UCP2 knockdown and genipin treatment mildly reduced glucose uptake, increased ROS production and decreased cell survival. Combining genipin and elesclomol further reduced glucose uptake and increased cellular and mitochondrial ROS production. Moreover, co-treatment with genipin and elesclomol reduced the colony forming capacity to 50.6±7.4% and the cell survival to 42.0±3.4% of that in the control cells (both P<0.001). Suppression of cell survival by treatment with elesclomol and genipin was enhanced in the presence of an exogenous ROS inducer and attenuated by a ROS scavenger. The cytotoxic effects of combining genipin and elesclomol were accompanied by reduced mitochondrial membrane potential and occurred through apoptosis as demonstrated by Annexin V assay and increased protein cleavage of PARP and caspase-3. Finally, in an A549 ×enograft mouse model, tumor growth was only modestly retarded by treatment with elesclomol or genipin alone, but was markedly suppressed by combining the two drugs compared with that in the control group (P=0.008). Therefore, high UCP2 expression may limit the antitumor effect of elesclomol by attenuating ROS responses, and this may be overcome by co-treatment with genipin; combining elesclomol and genipin may be an effective strategy for treating cancers with high UCP2.
RESUMEN
The aim of the present study was to investigate the metabolic and anticancer effects of troglitazone (TGZ) with a focus on the potential role of mitochondrial pyruvate utilization. 2Deoxyglucose (2DG) was more cytotoxic in CT26 cancer cells compared with T47D cells, despite a smaller suppression of glucose uptake. On the other hand, TGZ caused a more prominent shift to glycolytic metabolism and was more cytotoxic in T47D cells. Both effects of TGZ on T47D cells were dosedependently reversed by addition of methyl pyruvate (mPyr), indicating suppression of mitochondrial pyruvate availability. Furthermore, UK5099, a specific mitochondrial pyruvate carrier inhibitor, closely simulated the metabolic and antitumor effects of TGZ and their reversal by mPyr. This was accompanied by a substantial reduction of activated p70S6K. In CT26 cells, UK5099 did not reduce activated p70S6K and only modestly decreased cell proliferation. In these cells, combining glutamine restriction with UK5099 further increased glucose uptake and completely suppressed cell proliferation. Thus, TGZmediated inhibition of mitochondrial pyruvate utilization is an effective treatment for cancer cells that are more dependent on mitochondrial glucose metabolism. By contrast, cancer cells that are more glycolysisdependent may require suppression of glutamine utilization in addition to blocking mitochondrial pyruvate availability for a full antitumor effect.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Mitocondrias/metabolismo , Ácido Pirúvico/metabolismo , Troglitazona/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desoxiglucosa/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Glucólisis/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismoRESUMEN
We tested the hypothesis that tumor response to conventional bortezomib (BTZ) treatment is enhanced by targeted radiotherapy of resistant cancer stem cells (CSCs) that have characteristically poor proteasome function. This was accomplished by augmenting 131I uptake through expression of a sodium-iodide symporter (NIS) fusion protein that accumulates in cells with low proteasome activity. The NIS gene fused with the C-terminal of ornithine decarboxylase degron (NIS-cODC) was cloned. Stably expressing CT26/NIS-cODC cells and tumorsphere-derived CSCs were evaluated for NIS expression and radioiodine uptake. CT26/NIS-cODC cells implanted into mice underwent PET imaging, and tumor-bearing mice were treated with BTZ alone or with BTZ plus 131I. CT26/NIS-cODC cells accumulated NIS protein, which led to high radioiodine uptake when proteasome activity was inhibited or after enrichment for stemness. The cell population that survived BTZ treatment was enriched with CSCs that were susceptible to 131I treatment, which suppressed stemness features. Positron emission tomography and uptake measurements confirmed high 124I and 131I uptake of CT26/NIS-cODC CSCs implanted in living mice. In CT26/NIS-cODC tumor-bearing mice, whereas BTZ treatment modestly retarded tumor growth and increased stemness markers, combining 131I therapy suppressed stemness features and achieved greater antitumor effects. The NIS-cODC system offer radioiodine-targeted elimination of CSCs that are tolerant to proteasome inhibition therapy.
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Resistencia a Antineoplásicos/efectos de los fármacos , Radioisótopos de Yodo/administración & dosificación , Células Madre Neoplásicas/efectos de los fármacos , Ornitina Descarboxilasa , Simportadores , Animales , Bortezomib , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Radioisótopos de Yodo/metabolismo , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Inhibidores de ProteasomaRESUMEN
We investigated the relation of 99mTc-MIBI uptake to mitochondrial membrane potential (MMP) in cancer cell lines and patient-derived tumor cells (PDCs). In T47D and HT29 cells with low MDR1 expression, FCCP dose-dependently reduced MMP and 99mTc-MIBI accumulation in similar patterns with nearly perfect linear relationships. T47D and HT29 cells with high MDR1 expression had low 99mTc-MIBI accumulation that was minimally affected by FCCP dose. In these cells, verapamil markedly increased 99mTc-MIBI accumulation to magnitudes that were excessive compared to MMP increase. Decreased plasma membrane potential by verapamil and its recovery by FCCP suggested that enhanced 99mTc-MIBI transport through modified plasma membranes contributed to the excess accumulation. Evaluation of three different colon cancer PDCs with low to modest MDR1 expression verified that FCCP significantly suppressed MMP and similarly reduced 99mTc-MIBI accumulation. Verapamil partially recovered both MMP and 99mTc-MIBI accumulation that was lowered by FCCP. Importantly, a high linear correlation was found (r = 0.865) between 99mTc-MIBI accumulation and MMP in these cells. These findings indicate that low baseline 99mTc-MIBI uptake that is markedly increased by verapamil represents cancer cells with high levels of MDR1 expression. However, in cancer cells with low or modest levels of MDR1 expression that do not markedly increase 99mTc-MIBI uptake by verapamil, the magnitude of uptake is largely dependent on cellular MMP.
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Transporte Biológico/fisiología , Potencial de la Membrana Mitocondrial/fisiología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Tecnecio Tc 99m Sestamibi/metabolismo , Verapamilo/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Membrana Celular/metabolismo , Resistencia a Múltiples Medicamentos/fisiología , Células HT29 , Humanos , Radiofármacos/metabolismo , Células Tumorales CultivadasRESUMEN
UNLABELLED: There has been recent interest in the relationship between (18)F-FDG uptake and the angiogenic activity of endothelial cells (ECs). The angiogenic process is strongly dependent on the interaction of ECs with matrix fibronectin (FN), a key regulator of EC survival, migration, and proliferation. Therefore, we investigated how FN influences EC glucose uptake and elucidated the signaling pathways that mediate this effect. METHODS: Human umbilical vein ECs were allowed to adhere to FN-coated plates and were compared with control cells for (18)F-FDG uptake, membrane GLUT1 levels, and hexokinase activity. The roles of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), and Akt were evaluated with Western blotting, small interfering RNA (siRNA), and specific inhibitors. RESULTS: FN adhesion significantly enhanced the protein-corrected (18)F-FDG uptake in HUVEC, to 2.1-, 2.7-, and 4.3-fold that in control cells by 2, 3, and 5 d, respectively. This effect was mediated by the upregulation of both membrane GLUT1 expression and hexokinase activity and was accompanied by FAK activation. Silencing of FAK signaling by siRNA completely abrogated both FN-induced FAK phosphorylation and (18)F-FDG uptake. FN also activated PI3K and Akt, well-known angiogenesis mediators, and the inhibition of either pathway totally abolished the effect of FN on (18)F-FDG uptake. Nitric oxide, a downstream Akt effector that stimulates glucose uptake, was not involved in the metabolic effect of FN. CONCLUSION: The results of this study demonstrated that an EC-FN interaction induces strong enhancement of (18)F-FDG uptake through the upregulation of GLUT1 expression and hexokinase activity. The findings also showed that the response occurs through FAK-mediated activation of PI3K and Akt, indicating a role for this pathway in modulating EC glucose metabolism.