Quantitative analysis of chemotherapeutic effects in tumors using in vivo staining and correlative histology.

AIMS: To microscopically analyze the chemotherapeutic response of tumors using in vivo staining based on an annexinV-Cy5.5 probe and independently asses their apoptotic count using quantitative histological analysis. METHODS: Lewis Lung Carcinomas cells, that are sensitive (CS-LLC) and resistant (CR-LLC) to chemotherapy were implanted in nude mice and grown to tumours. Mice were treated with cyclophosphamide and injected with a Cy5.5-annexinV fluorescent probe. In vivo imaging was performed using Fluorescence Molecular Tomography. Subsequently tumours were excised and prepared for histology. The histological tumour sections were stained for apoptosis using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. A minimum of ten tissue sections were analyzed per tumour for apoptosis quantification by TUNEL staining and corresponding Cy5.5 distribution. RESULTS: We detected higher levels of apoptosis and corresponding higher levels of Cy5.5 fluorescence in the CS-LLC vs. the CR-LLC tumours. The cell count rate on CS-LLC sections over CR-LLC was found to be approximately 2 :1 where the corresponding area observed on Cy5.5 distribution measurements revealed a approximately 1.7 :1 ratio of CS-LLC over CR-LLC. These observations are consistent with the higher apoptotic index expected from the CS-LLC cell line. CONCLUSIONS: Quantitative analysis of histological slices revealed higher fluorescence and higher apoptotic count in the CS-LLC tumour images compared to the CR-LLC tumour images. These observations demonstrate that the annexinV-Cy5.5 probe sensed the chemotherapeutic effect of cyclophospamide and further confirmed in vivo FMT measurements.

Imaging apoptosis in vivo using 124I-annexin V and PET.

Abnormal regulation of apoptosis is an important pathogenic mechanism in many diseases including cancer. Techniques to assess apoptosis in living organisms are limited and, in the case of solid organs, restricted to histological examination of biopsy samples. We investigated the use of (124)I-annexin V, which binds to phosphatidylserine (PS) on the surface of apoptotic cells, as a potential positron emission tomography (PET) radioligand for the noninvasive measurement of apoptosis in vivo. Annexin V and a similar-sized protein, ovalbumin, were directly labelled with (124)I. We report the validation of (124)I-annexin V in vitro and in an animal model of liver apoptosis that has not previously been used to test iodinated annexin V. Also, for the first time, we report metabolite analysis of (124)I-annexin V and the correlation of (124)I-annexin V uptake with apoptotic density (AD). Sixfold more (124)I-annexin V was associated with Jurkat cells after apoptosis induction, indicating that PS binding by annexin V was preserved after iodination. (124)I-ovalbumin did not demonstrate increased uptake in apoptotic cells. In normal BDF-1 mice, the radioligand was rapidly cleared, but some in vivo dehalogenation resulted in the accumulation of activity in the thyroid and stomach content. PET images demonstrated uptake of (124)I-annexin V but not (124)I-ovalbumin in apoptotic liver lesions. In vivo (124)I-annexin V uptake, derived from PET images, correlated with histologically derived AD (r=.86, P<.01). These results demonstrate that (124)I-annexin V is localised to anti-Fas-induced apoptosis, in contrast to (124)I-ovalbumin, which did not show preferential uptake in the apoptotic liver.

Functional comparison of annexin V analogues labeled indirectly and directly with iodine-124.

We are interested in imaging cell death in vivo using annexin V radiolabeled with (124)I. In this study, [(124)I]4IB-annexin V and [(124)I]4IB-ovalbumin were made using [(124)I]N-hydroxysuccinimidyl-4-iodobenzoate prepared by iododestannylation of N-hydroxysuccinimidyl-4-(tributylstannyl)benzoate. [(124)I]4IB-annexin V binds to phosphatidylserine-coated microtiter plates and apoptotic Jurkat cells and accumulates in hepatic apoptotic lesions in mice treated with anti-Fas antibody, while [(124)I]4IB-ovalbumin does not. In comparison with (124)I-annexin V, [(124)I]4IB-annexin V has a higher rate of binding to phosphatidylserine in vitro, a higher kidney and urine uptake, a lower thyroid and stomach content uptake, greater plasma stability and a lower rate of plasma clearance. Binding of radioactivity to apoptotic cells relative to normal cells in vitro and in vivo appears to be lower for [(124)I]4IB-annexin V than for (124)I-annexin V.

Evaluation of 18F-annexin V as a PET imaging agent in an animal model of apoptosis.

UNLABELLED: Annexin V is a 36-kDa protein that binds with high affinity to phosphatidylserine lipids in the cell membrane. Because one of the earliest measurable events in apoptosis is the eversion of phosphatidylserine from the inner membrane leaflet to the outer cell surface, annexin V has proven useful for detecting the earliest stages of apoptosis. METHODS: Annexin V was radiolabeled with 18F using N-succinimidyl-4-18F-fluorobenzoic acid chemistry, to a specific activity of 555-925 kBq/mug of protein. 18F-Annexin V (14.8-51.8 MBq) was administered intravenously to rats after pretreatment with cycloheximide (5 mg/kg) to induce liver apoptosis, and the injected rats were imaged by PET over 2 h. After imaging, rats were dissected and individual organs were weighed and counted. RESULTS: Pretreatment of rats with cycloheximide resulted in a 3- to 9-fold increase in uptake of 18F-annexin V in the liver of treated animals at 2 h, compared with controls. By morphologic analysis, treated livers showed a 3- to 6-fold higher level of apoptosis than controls, with higher levels also seen with longer exposure to cycloheximide. Terminal deoxynucleotide end-labeling (TUNEL) assays performed on liver slices showed that cycloheximide induced a 5- to 8-fold increase in the number of TUNEL-positive nuclei. These TUNEL results correlated with the uptake of 18F-annexin V in dissected liver tissue, with an r2 value of 0.89. Biodistribution analysis of normal rats showed highest uptake of 18F-annexin V in the kidneys and urinary bladder, indicating rapid renal clearance of 18F-annexin V metabolites. CONCLUSION: The PET data, the organ-specific uptake data from dissection, and the morphologic and TUNEL measures of apoptosis together indicate that 18F-annexin V binds specifically to apoptotic tissues in this model of chemically induced apoptosis in rat liver. The short physical half-life of 18F-annexin V and the rapid clearance of its metabolites to the urinary system suggest that 18F-annexin V will be useful in early assessment of the clinical response to cancer therapy in individual patients.

Effects of ibuprofen and hypoxia on neutrophil apoptosis in neonates.

BACKGROUND: Ibuprofen is a cyclooxygenase inhibitor that is effective in treating patent ductus arteriosus in preterm infants. However, recent trials have suggested that it may increase the risk of developing necrotizing enterocolitis and chronic lung disease. Apoptosis of neutrophils is impaired in newborns, leading to reduced clearance of activated cells and possibly contributing to the susceptibility of infants to these inflammatory diseases. OBJECTIVES: In the present studies, we investigated the hypothesis that ibuprofen reduces neonatal neutrophil apoptosis in the setting of hypoxia or lipopolysaccharide (LPS). METHODS: Neutrophils and peripheral blood mononuclear cells were isolated from adult and cord blood and cultured in the presence or absence of ibuprofen (1.5 mM), hypoxia (<5% O2), and bacterial LPS (100 ng/ml). Apoptosis was quantified by measuring binding of FITC-Annexin V using flow cytometry. Cytokine concentrations in cell supernatants were measured by ELISA. RESULTS: After 24 h, 20% of adult and 14% of neonatal neutrophils were apoptotic. Apoptosis was reduced by hypoxia in both adult and neonatal cells. Ibuprofen further reduced neutrophil apoptosis, but only when the cells were cultured in the presence of mixed leukocytes. This suggests that the effects of ibuprofen on apoptosis are dependent on soluble products secreted by peripheral blood mononuclear cells. We found that production of tumor necrosis factor (TNF)-alpha by neonatal leukocytes was significantly increased by ibuprofen, and was further increased following exposure to ibuprofen in the presence of LPS and hypoxia. In contrast, production of macrophage inflammatory protein (MIP)-1alpha was not affected by treatment with ibuprofen, and ibuprofen blocked induction of this chemokine by LPS. CONCLUSION: We conclude that the net effect of ibuprofen on neutrophils is antiapoptotic, especially in the presence of hypoxia or LPS. This effect may be mediated, in part, by increased production of TNF-alpha by peripheral blood mononuclear cells. These data suggest that treatment of infants with ibuprofen, in the presence of infection and/or tissue hypoperfusion/hypoxia, may contribute to the development of inflammatory diseases.

Preparation of F-18 labeled annexin V: a potential PET radiopharmaceutical for imaging cell death.

The clinical response to antitumor therapy is measured using imaging, such as CT or MRI, 6-12 weeks following chemotherapy treatment. The images at that time reflect both tumor cell death and new growth. Therefore, the amount of tumor cell death caused by chemotherapy cannot be efficiently quantified with current imaging modalities. A quantitative measurement of tumor cell death immediately following chemotherapy is needed to help validate both new agents and to optimize administration of existing therapies. Annexin V is a 36kD protein that binds to exposed phosphatidylserine (PS) on dying cells. In order to synthesize a probe that can detect cell death in vivo, the positron emitter F-18 was conjugated to annexin V via the compound N- succinimidyl-4-[18F]fluorobenzoate, [18F]SFB. The decay corrected radiochemical yield of F-18 labeled annexin V from 18F fluoride was 17.6 +/- 5.6% (n = 4) in three hours. The stepwise radiochemical yield of the conjugation step with annexin V was as high as 70% when a protein concentration of 5 mg/ml was used. Cancer cells treated with the chemotherapeutic agent, etoposide, showed an 88% increase in the binding of F-18 labeled annexin V compared to untreated cells. We conclude that [18F] labeled annexin V can be readily prepared by the conjugation of annexin V with [18F]SFB and that the positron-emitting compound is biologically active in detecting apoptosis.

Damage to F-actin and cell death induced by chromium VI and nickel in primary monolayer cultures of rat hepatocytes.

The toxicity of hexavalent chromium and nickel was investigated using primary cultures of hepatocytes as an in vitro system. Cr VI and Ni are widely used in the steel and orthopaedic implant industry. Although their toxicity has been extensively investigated, the mechanism(s) of action is/are not fully understood. Monolayer cultures of hepatocytes (10(5) cells/cm2) were exposed to various concentrations of Cr VI and Ni for 24 h. Cells were stained with phalloidin-FITC for the detection of the cytoskeletal component, F-actin, and Annexin V-FITC and propidium iodide for the detection of the mode of cell death. Exposure of cells to Cr VI (1, 5, 10 and 50 microM) resulted in the loss of the cell cytoskeleton, and this was accompanied by membrane blebbing and shrinking of the cell. Ni, on the other hand, induced detectable damage to the cytoskeleton only at 500 microM. Staining of the cells with Annexin V and propidium iodide showed that Cr VI induces apoptosis at low concentrations (5 microM), and necrosis at higher concentrations (25 and 50 microM). Ni almost exclusively induced necrosis at 500 microM with very few cells undergoing apoptosis. Below this concentration it had no discernable effect on hepatocytes. Damage to the cell cytoskeleton caused by Cr VI may be an early indication of apoptosis in hepatocytes.

The ApoCorrect assay: a novel, rapid method to determine the biological functionality of radiolabeled and fluorescent Annexin A5.

We have demonstrated that imaging of programmed cell death (PCD) in patients is possible using 99mTc-Annexin A5. Because of the short half-life of the technetium label it is important to limit the time span between the preparation of 99mTc-Annexin A5 and its administration into the patient. Therefore methods of quality control that determine the biological active fraction in the 99mTc-Annexin A5 should be not only accurate and precise but also rapid. We report the development and validation of a rapid, simple assay measuring the biological active fraction of 99mTc-Annexin A5. The assay is based on a solid phase of paramagnetic beads which are coated with phospholipids. Annexin A5 binds to these beads with high affinity if phosphatidyl serine is present within the phospholipid coat. Furthermore the binding depends on Ca2+ ions and functional Ca2+/phospholipid binding sites of Annexin A5. The bead assay is specific, stability-indicating, repeatable, and reproducible. It allows one to determine within 25 min the biological active fraction of a 99mTc-Annexin A5 preparation. We dubbed this assay the ApoCorrect assay.

Imaging taxane-induced tumor apoptosis using PEGylated, 111In-labeled annexin V.

UNLABELLED: 99mTc-Labeled annexin V has been used for the imaging of tumor apoptosis induced by chemotherapy. However, owing to the short half-life of annexin V, multiple injections of the radiotracer are necessary to capture the peak apoptotic activity. In this study, we evaluated the imaging properties of an (111)In-labeled, long-circulating annexin V. METHODS: Both polyethylene glycol (PEG) and the metal chelator diethylenetriaminepentaacetic acid (DTPA) were simultaneously introduced to annexin V or ovalbumin through the use of a heterofunctional PEG precursor. Imaging studies were performed in mice bearing subcutaneously inoculated human mammary MDA-MB-468 tumors. The mice were treated with poly(L-glutamic acid)-paclitaxel, monoclonal antibody C225, or a combination of poly(L-glutamic acid)-paclitaxel and C225, followed by intravenous injection of (111)In-DTPA-PEG-annexin V. Images were acquired 48 h after the injection of the radiotracer. Autoradiography and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) staining were performed on adjacent tumor slices for the localization of apoptotic cells. The imaging properties of unPEGylated annexin V and PEGylated ovalbumin were also determined to permit assessment of the specificity of (111)In-DTPA-PEG-annexin V. RESULTS: Tumor apoptotic index increased from 1.67% +/- 0.31% at baseline to 7.60% +/- 0.72% and 11.07% +/- 1.81%, respectively, 4 d after treatment with poly(L-glutamic acid)-paclitaxel or combined poly(L-glutamic acid)-paclitaxel and C225. Tumor uptake (percentage of injected dose per gram of tumor [%ID/g]) of PEGylated (111)In-DTPA-PEG-annexin 4 d after treatment was significantly higher in tumors treated with poly(L-glutamic acid)-paclitaxel (10.76 +/- 1.38 %ID/g; P = 0.001) and with combined poly(L-glutamic acid)-paclitaxel and C225 (9.84 +/- 2.51 %ID/g; P = 0.029) than in nontreated tumors (6.14 +/- 0.67 %ID/g), resulting in enhanced visualization of treated tumors. (111)In-DTPA-PEG-annexin V distributed into the central zone of tumors, whereas (111)In-DTPA-annexin V was largely confined to the tumor periphery. Furthermore, uptake of (111)In-DTPA-PEG-annexin V by tumors correlated with apoptotic index (r = 0.87, P = 0.02). Increase in tumor uptake of the nonspecific PEGylated protein (111)In-DTPA-PEG-ovalbumin was also observed after poly(L-glutamic acid)-paclitaxel treatment (55.6%), although this increase was less than that observed for (111)In-DTPA-PEG-annexin V (96.7%). CONCLUSION: Increased uptake of and improved visualization with (111)In-DTPA-PEG-annexin V in solid tumors after chemotherapy are mediated through both specific binding to apoptotic cells and nonspecific retention of macromolecular contrast agents in the tumors. (111)In-Labeled, PEGylated annexin V may be used to assess tumor response to chemotherapy.

Detection of phosphatidylserine surface exposure on human erythrocytes using annexin V-ferrofluid.

The asymmetric transbilayer distribution of phospholipids in the plasma membrane and the regulation of phosphatidylserine (PS) exposure at the cell surface of animal cells are of high physiological significance. It has been shown previously that annexin V is one of the most sensitive tools with which the presence of small amounts of PS on the outer surface of eukaryotic cells can be detected. We present here the covalent coupling of annexin V molecules to magnetic nanoparticles of maghemite. The resulting annexin V-ferrofluid is used in the magnetic separation of PS exposing cells, as illustrated for human erythrocytes modified in their phospholipid transbilayer asymmetry by the use of a calcium ionophore. Results on stored human erythrocytes and comparison with results obtained using iodinated and fluorescein-labeled annexin V are also presented.