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• W2089986662 abstract "Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by NG-nitro-l-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only ∼55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis. Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by NG-nitro-l-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only ∼55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis. In the lung, metastatic neoplasms are the most common malignant tumors. Their formation usually involves hematogenous seeding of metastatic cancer cells into the lung from remote primary tumors. Interactions between intravascular cancer cells and the endothelium are important determinants of metastatic outcome.1Al Mehdi AB Tozawa K Fisher AB Shientag L Lee A Muschel RJ Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.Nat Med. 2000; 6: 100-102Crossref PubMed Scopus (559) Google Scholar, 2Orr FW Wang HH Tumor cell interactions with the microvasculature: a rate-limiting step in metastasis.Surg Oncol Clin N Am. 2001; 10: 357-381Abstract Full Text PDF PubMed Google Scholar For example, the expression of constitutive and inducible microvascular adhesion molecules, and the release of reactive oxygen species (NO, O2−, and H2O2) by endothelial cells or cancer cells can regulate the mechanisms that govern the metastatic process, including cancer cell adhesion and arrest,3Scherbarth S Orr FW Intravital videomicroscopic evidence for regulation of metastasis by the hepatic microvasculature: effects of interleukin-1alpha on metastasis and the location of B16F1 melanoma cell arrest.Cancer Res. 1997; 57: 4105-4110PubMed Google Scholar the production of endothelial matrix metalloproteinases,4Belkhiri A Richards C Whaley M McQueen SA Orr FW Increased expression of activated matrix metalloproteinase-2 by human endothelial cells after sublethal H2O2 exposure.Lab Invest. 1997; 77: 533-539PubMed Google Scholar and cancer cell apoptosis.5Wang HH McIntosh AR Hasinoff BB Rector ES Ahmed N Nance DM Orr FW B16 melanoma cell arrest in the mouse liver induces nitric oxide release and sinusoidal cytotoxicity: a natural hepatic defense against metastasis.Cancer Res. 2000; 60: 5862-5869PubMed Google Scholar Evidence from in vitro and in vivo studies has shown that reactive oxygen and nitrogen species can be cytotoxic to neoplastic cells6Li LM Kilbourn RG Adams J Fidler IJ Role of nitric oxide in lysis of tumor cells by cytokine-activated endothelial cells.Cancer Res. 1991; 51: 2531-2535PubMed Google Scholar, 7Anasagasti MJ Alvarez A Martin JJ Mendoza L Vidal-Vanaclocha F Sinusoidal endothelium release of hydrogen peroxide enhances very late antigen-4-mediated melanoma cell adherence and tumor cytotoxicity during interleukin-1 promotion of hepatic melanoma metastasis in mice.Hepatology. 1997; 25: 840-846Crossref PubMed Scopus (55) Google Scholar, 8Jessup JM Battle P Waller H Edmiston KH Stolz DB Watkins SC Locker J Skena K Reactive nitrogen and oxygen radicals formed during hepatic ischemia-reperfusion kill weakly metastatic colorectal cancer cells.Cancer Res. 1999; 59: 1825-1829PubMed Google Scholar, 9Carretero J Obrador E Esteve JM Ortega A Pellicer JA Sempere FV Estrela JM Tumoricidal activity of endothelial cells. Inhibition of endothelial nitric oxide production abrogates tumor cytotoxicity induced by hepatic sinusoidal endothelium in response to B16 melanoma adhesion in vitro.J Biol Chem. 2001; 276: 25775-25782Crossref PubMed Scopus (47) Google Scholar and reduced their adhesion to postcapillary venules.10Kong L Dunn GD Keefer LK Korthuis RJ Nitric oxide reduces tumor cell adhesion to isolated rat postcapillary venules.Clin Exp Metastasis. 1996; 14: 335-343Crossref PubMed Scopus (49) Google Scholar In vivo, we have recently demonstrated that the arrest of intravascular B16F1 melanoma cells in the liver induces the rapid local release of nitric oxide (NO) that causes apoptosis of the melanoma cells and inhibits their subsequent development into hepatic metastases.5Wang HH McIntosh AR Hasinoff BB Rector ES Ahmed N Nance DM Orr FW B16 melanoma cell arrest in the mouse liver induces nitric oxide release and sinusoidal cytotoxicity: a natural hepatic defense against metastasis.Cancer Res. 2000; 60: 5862-5869PubMed Google Scholar Because pulmonary endothelial cells generate NO in response to shear stress,11Fukaya Y Ohhashi T Acetylcholine- and flow-induced production and release of nitric oxide in arterial and venous endothelial cells.Am J Physiol. 1996; 270: H99-H106PubMed Google Scholar we have postulated that there is a comparable cytotoxic mechanism in the lung. Here we provide data showing that the arrest of B16F1 melanoma cells in the pulmonary circulation of wild-type C57B1/6 mice (WT mice) induces the local release of NO that is endothelial nitric oxide synthase (eNOS)-dependent. In turn NO causes the melanoma cells to undergo apoptosis and inhibits their development into pulmonary metastatic tumors. The existence of comparable mechanisms in the lung and the liver suggests that NO may be a natural defense against the formation of metastatic tumors in organs with complex microvascular networks. Fluoresbrite carboxylate YG microsphere-labeled B16F1 melanoma cells were injected into the tail veins of C57Bl/6 mice. The lungs were removed between 0 and 48 hours after cell injection to measure NO production, tumor cell apoptosis, and the clearance of tumor cells from the lung. To study metastasis, unlabeled B16F1 cells were injected and the lungs were collected 7 or 14 days later to count surface metastatic nodules or to quantify metastasis by histomorphometry. All analyses were performed blindly. WT female C57Bl/6 mice, weighing 20 to 22 g, were purchased from Charles River Breeding Laboratories (Montreal, QC, Canada or Kingston, NY) and housed according to University of Manitoba guidelines. eNOS knockout C57Bl/6 mice (eNOS KO mice), age 5 to 6 weeks, were obtained from Jackson Laboratory (Bar Harbor, ME). The murine B16F1 melanoma cell line was obtained from the American Type Culture Collection (Rockville, MD). Fluorescent polystyrene microspheres (15 μm in diameter) were purchased from Bangs Laboratories, Inc. (Fisher, IN). Fluoresbrite carboxylate YG microspheres (0.05 μm) were purchased from Polysciences, Inc. (Warrington, PA) and MitoTracker Red from Molecular Probes (Eugene, OR). α-MEM, Opti-MEM reduced serum medium, penicillin-streptomycin, and trypsin-ethylenediaminetetraacetic acid were purchased from Life Technologies (Burlington, ON, Canada). Avertin (2,2,2-tribromoethanol), diethyldithiocarbamate (DETC), and NG-nitro-l-arginine methyl ester (L-NAME) were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). 4,5-Diaminofluorescein diacetate (DAF-2 DA) was obtained from Calbiochem (La Jolla, CA). The ApopTag Peroxidase in Situ Apoptosis Detection Kit S7100 was from Intergen Company (Purchase, NY). B16F1 cells were cultured in α-MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution, in a T25 culture flask (Corning Glass, Corning, NY) to 95% confluence. The culture medium was then replaced by Opti-MEM serum-reduced medium and fluoresbrite carboxylate microspheres were added to the flask in a ratio of 100 μl of microspheres per 5 ml of Opti-MEM medium (microsphere stock, 2.5% solids-latex). The flask was gently mixed and put back in the incubator for 2 hours to label the cells with occasional shaking of the flask. The cells were rinsed three times with Opti-MEMI medium to wash off the unincorporated beads and left in α-MEM culture medium overnight to reduce cell aggregation on detaching. The labeled cells were detached on the following day with 0.5× trypsin-ethylenediaminetetraacetic acid at 37°C for 5 minutes. The cell suspension was centrifuged at 170 × g for 3 minutes. The cell pellet was resuspended in pyrogen-free saline and kept on ice before injection. Cells with a viability >95%, measured by trypan blue exclusion, were used in the study. Mice were anesthetized with avertin (30 mg/ml; 0.2 to 0.3 ml/mouse, IP). A fluorescent-labeled cell suspension of 5 × 105 B16F1 melanoma cells in a volume of 150 μl of saline was injected into the tail vein throughout 2 to 3 minutes, using a 30G1/2 needle and 1-ml syringe. The lung was collected under anesthesia at specific times (0 to 48 hours) after injection. For 0-hour samples, the lung tissue was removed immediately at the end of the injection (within 30 seconds to 1 minute). To determine whether the shear stress of tumor cells may also play a role in the induction of NO by lung microvascular endothelial cells, we injected 5 × 105 15-μm fluorescent-labeled polystyrene microspheres in a volume of 150 μl of saline intravenously. The lung tissue was collected 20 minutes afterward for electron paramagnetic resonance (EPR) measurement. Based on our previous study,5Wang HH McIntosh AR Hasinoff BB Rector ES Ahmed N Nance DM Orr FW B16 melanoma cell arrest in the mouse liver induces nitric oxide release and sinusoidal cytotoxicity: a natural hepatic defense against metastasis.Cancer Res. 2000; 60: 5862-5869PubMed Google Scholar the competitive NO synthase inhibitor L-NAME was administered to inhibit NO production and to test its effects on cell clearance, apoptosis, and metastasis formation. For cell clearance and apoptosis analysis, 5 mg/kg of L-NAME was injected intraperitoneally 1 hour before cell injection and 2.5 mg/kg of L-NAME was co-injected with the cells. For the metastasis assays, 2.5 mg/kg of L-NAME was co-injected with the cells and 5 mg/kg of L-NAME was injected intraperitoneally 20 hours after the cell injection. EPR spectroscopy was used to quantitatively measure the production of NO. The NO trapping agents DETC (400 mg/kg in saline, IP) and FeSO4/sodium citrate (40 mg/kg and 200 mg/kg, respectively, mixed in water, SC) were given to each mouse 30 minutes before obtaining the lung sample. The mice were anesthetized 10 minutes before sacrifice. The thorax was opened and the lungs were removed (between 30 seconds to 1 minute) and placed onto a precooled Petri dish on ice. The lungs were quickly sliced into smaller pieces, placed into a precooled 1-ml syringe, and transferred into a Suprasil synthetic quartz tube (2.4-mm inner diameter; Heraeus Amersil, Duluth, GA) by pushing the tissue gently through the syringe to fill the tube up to 3 to 4 cm in height. The tube was then immediately placed into liquid nitrogen until NO was measured by EPR spectroscopy.12Doi K Akaike T Horie H Noguchi Y Fujii S Beppu T Ogawa M Maeda H Excessive production of nitric oxide in rat solid tumor and its implication in rapid tumor growth.Cancer. 1996; 77: 1598-1604Crossref PubMed Scopus (0) Google Scholar EPR spectroscopy, used for the measurement of trapped NO-Fe2+-(DETC)2 complex, was performed at 125 K (temperature controller model BVT-3000; Bruker Spectrospin Ltd., Karlsruhe, Germany). The spectra were measured with a Bruker model EMX EPR X-band spectrometer system operating at 9.25 GHz with 100 kHz modulation. The instrument settings were as follows: 1) microwave power, 5 mW; 2) modulation amplitude, 5 G and signal level, 1 × 103; and 3) scan range, 500 G. The concentration of the NO-Fe2+-(DETC)2 complex in each sample was assumed to be proportional to the signal amplitude (peak-to-trough) of the triplet-hyperfine structure (hyperfine splitting of 13 G) observed at g = 2.04 (see below). Data were expressed as relative EPR signal intensities (arbitrary units) after subtracting the Cu2+-(DETC)2 complex signal observed in all samples. As described in our previous work,5Wang HH McIntosh AR Hasinoff BB Rector ES Ahmed N Nance DM Orr FW B16 melanoma cell arrest in the mouse liver induces nitric oxide release and sinusoidal cytotoxicity: a natural hepatic defense against metastasis.Cancer Res. 2000; 60: 5862-5869PubMed Google Scholar we performed a double-integration calibration to calibrate the conversion from peak-to-peak EPR amplitudes to nmol ON-Fe2+-(DETC)2/gram of wet tissue concentrations (see the caption to Figure 1). The only difference is that we used the Bruker EPR spectrometer for our present work, whereas the Varian E-12 spectrometer was used previously.5Wang HH McIntosh AR Hasinoff BB Rector ES Ahmed N Nance DM Orr FW B16 melanoma cell arrest in the mouse liver induces nitric oxide release and sinusoidal cytotoxicity: a natural hepatic defense against metastasis.Cancer Res. 2000; 60: 5862-5869PubMed Google Scholar We have performed EPR measurements on the same samples at 125 K on both the Varian and Bruker EPR systems to relate the peak height measurements from one instrument to the other. AWIN-EPR software (Bruker Spectrospin Ltd.) was used for nearly all EPR data manipulation on a Compaq Deskpro P500 computer (Compaq Computer Corp., Houston, TX). NO generation was monitored in situ by labeling the pulmonary endothelium with DAF-2 DA that is de-esterified intracellularly to DAF-2. NO and its higher oxides, such as NOx or nitrous anhydride (N2O3), provide the third nitrogen to form a triazo ring from the two amino groups of the nonfluorescent DAF-2 and convert it to diaminotriazolofluorescein (DAF-2T) that is fluorescent and can be monitored at 490 nm excitation and 530 nm emission.13Al Mehdi AB Song C Tozawa K Fisher AB Ca2+- and phosphatidylinositol 3-kinase-dependent nitric oxide generation in lung endothelial cells in situ with ischemia.J Biol Chem. 2000; 275: 39807-39810Crossref PubMed Scopus (38) Google Scholar We used an established intact organ microscopy technique to image microvascular endothelial cells in situ in isolated, ventilated, blood-free mouse lungs in real time using an epifluorescence microscope.14Al Mehdi AB Zhao G Dodia C Tozawa K Costa K Muzykantov V Ross C Blecha F Dinauer M Fisher AB Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+.Circ Res. 1998; 83: 730-737Crossref PubMed Scopus (248) Google Scholar Briefly, female WT mice, weighing 20 to 22 g were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg). A tracheostomy was performed, and artificial ventilation with 95% air and 5% CO2 (BOC Group, Inc., Murray Hill, NJ) was started through a cannula. The abdomen was opened and the animal was exsanguinated by transection of the major abdominal vessels. A cannula was inserted into the main pulmonary artery via a puncture in the right ventricle, and another was inserted into the left atrium. The lung was cleared of blood by gravity perfusion via the pulmonary artery with an artificial medium (Krebs-Ringer bicarbonate buffer KRB: NaCl, 118.45; KCl, 4.74; MgSO4/7H2O, 1.17; CaCl2/2H2O, 1.27; KH2PO4, 1.18; and NaHCO3, 24.87, in mmol/L, with 5% dextran and 10 mmol/L glucose at pH 7.4). The flow-through perfusate left the lung via the left atrial cannula. Once the lung became visibly cleared of blood, the heart-lung preparation was dissected en bloc and was placed onto a 48 × 60 × 0.16-mm coverglass window in a specially designed Plexiglas chamber with ports for the tracheal, pulmonary, and left atrial cannulas. The cardiovascular ports were connected to a peristaltic pump that recirculated 30 ml of perfusate at a constant flow rate of 2 ml/min through the pulmonary vascular bed. The chamber was placed on the stage of an epifluorescence microscope fitted with a ×60 objective (Nikon Diaphot TMD) and equipped with an optical filter changer (Lambda 10-2; Sutter Instrument Co., Novato, CA). A local anesthetic (0.05 mg of xylazine, Sigma) was injected subepicardially into the posterior wall of the right atrium to abolish lung movement artifact because of contraction of remaining cardiac muscle. Excitation of the lung surface was accomplished with a mercury lamp fiber optic light source, a fluorescein isothiocyanate filter set for DAF-2T (HQ41001 with 480/40 excitation filter, 505 LP dichroic mirror, and 535/40 emission filter; Chroma Technology Corp., Brattleboro, VT). The integrity of the preparation was continuously monitored by online measurements of intratracheal and pulmonary artery perfusion pressures. Endothelial cells in the subpleural vasculature were positively identified by labeling with DiI-acetylated low-density lipoprotein added to the perfusate (a tetramethylrhodamine isothiocyanate filter set for DiI-acetylated low-density lipoprotein, Chroma Technology Corp.). We used a Nikon Diaphot TMD epifluorescence microscope, a Hamamatsu ORCA-100 digital camera (Hamamatsu Corp., Bridgewater, NJ), and MetaMorph imaging software (Universal Imaging Corp., Downingtown, PA) for imaging. After an equilibration period of 45 minutes with the isolated lung to allow uptake of DAF-2 DA (5 μmol/L), intravascular dye was removed by perfusion with dye-free medium for 5 minutes to reduce background fluorescence. Mitotracker-Red labeled B16F1 cells (2 × 106) were injected through a pulmonary artery port by peristaltic pump at 2 ml/min. Images of DAF-2T-stained vascular endothelial cells were taken from the same area every 3 seconds for up to 20 minutes during which ventilation was stopped. As a control, after the equilibration period, images were taken during continuous perfusion. For quantification, each endothelial cell was outlined and its fluorescence intensity was measured throughout time. For each lung, mean fluorescence intensities of four to seven endothelial cells were averaged. Fluorescence intensity of three to four lungs for each condition was averaged. Data are expressed as relative fluorescence intensities (arbitrary units). All results are expressed as mean ± SE for each condition. Lung tissues for in situ DNA end-labeling were collected at 0, 4, 8, 24, and 48 hours after tail vein injection of fluorescent-labeled B16F1 cells. No trapping agent was given to these animals. For each time point five animals were used. Frozen sections (12 μm) were obtained from the upper and median lobes of the right lung and upper lobe of the left lung. In situ DNA end labeling using a digoxigenin-peroxidase detection system (ApopTag S7100 kit, Intergen) was performed on these sections that contained injected fluorescent-labeled melanoma cells. To achieve optimal double labeling, the procedures were performed according to the manufacture's instructions with the following modifications, as previously described.5Wang HH McIntosh AR Hasinoff BB Rector ES Ahmed N Nance DM Orr FW B16 melanoma cell arrest in the mouse liver induces nitric oxide release and sinusoidal cytotoxicity: a natural hepatic defense against metastasis.Cancer Res. 2000; 60: 5862-5869PubMed Google Scholar 1) Proteinase K digestion (20 μg/ml) was added before postfixing the sections with ethanol/acetic acid (2:1, v/v), to facilitate the unmasking of fragmented DNA in apoptotic tumor cells. 2) Incubation for in situ DNA end labeling reaction was performed overnight in a humidified chamber at 37°C, with subsequent 1 hour anti-digoxigenin peroxidase conjugate incubation at room temperature in a humidified chamber and 12 minutes peroxidase substrate color development. 3) The counter stain specimen and mount specimen steps were omitted to prevent the quenching of fluorescence in the microspheres by organic solvents. 4) The slides were mounted with Gel/Mount (Biomeda Corp, Foster City, CA). To generate positive control samples for apoptosis, labeled B16F1 cells were exposed to 2 μm of NO donor S-nitroso-N-acetyl-penicillamine (SNAP) for 24 hours. Detached and collected cell pellets were resuspended in 1% paraformaldehyde at 1 × 106/150 μl of saline. Lung tissues were collected immediately after injection and fixed in 1% paraformaldehyde. The total number of both single fluorescent- and double-labeled cells (fluorescent/ApopTag DNA end-labeling) was scored using a fluorescence microscope with dual illumination of the fluorescence and incandescence (×200 magnification). More than 100 total cells from both left and right lobes were counted for most samples. The percentage of in situ DNA end-labeled melanoma cells was calculated by the formula: in situ end-labeled tumor cells (%) = 100 × [number of double-labeled cells]/[total number of cells (double labeled + single labeled)]. Frozen sections (12 μm) were obtained from the upper and median lobes of the right lung and the upper lobe of the left lung, collected at 0, 4, 8, 24, and 48 hours as described above. Fluorescent-labeled B16F1 cell fragments and the intact fluorescent-labeled B16F1 melanoma cells in 100 microscopic fields (×200 magnification) were counted from each lobe to determine the number of B16F1 cells in the lung at each time point. The ratio of cell fragments to intact cells at 8 hours and 48 hours was calculated as follows: cell fragments (%) = (cell fragments/cell fragments + intact cells) × 100. Data were expressed as the mean of five values. Unlabeled B16F1 cells (5 × 105/150 μl saline) were injected into the tail vein of the mice and allowed to grow in vivo for 7 or 14 days. All animal surgical procedures were approved by the Bannatyne Campus Protocol Management and Review Committee at the University of Manitoba. All animal care was given according to the guidelines of the Central Animal Care Services at the University of Manitoba. The mice were sacrificed under anesthesia and the lungs were fixed in 10% neutral buffered formalin. All metastatic nodules identifiable on the surface of all lung lobes were counted using a dissecting microscope on the day 7 after tumor cell injection. Fourteen days after injection, histomorphometric analysis was performed on histological sections from all five lobes using a Merz Graticule to quantify the percentage of tissue area occupied by the metastases. Prism software was used for statistical analysis. For EPR, tumor cell apoptosis and clearance, an unpaired t-test was used. The Mann-Whitney U-test was used for metastasis studies. Comparisons of DAF-2T fluorescence intensity were made using analysis of variance with Bonferroni's test using SigmaPlot 2000 (SPSS Inc., Chicago, IL). To determine whether NO is released in the lung after B16F1 cell arrest, we injected 5 × 105 B16F1 cells, in a volume of 150 μl of saline, into the lateral tail vein of the WT C57Bl/6 mice. We injected the NO spin trap DETC/FeSO4 30 minutes before harvesting the tissue to detect NO generation by EPR spectroscopy. As shown in Figure 1, NO generation was induced as soon as tissue could be harvested after cell injection, increased sevenfold within 20 minutes after cell injection compared to the saline control at 0 minutes, and approached basal levels by 4 hours. Rapid and significant production of NO was also detected in mice injected with 5 × 105 fluorescent microspheres with similar diameter to that of B16F1 cells (16 ± 4 μm). In these animals, the peak NO level was significantly lower than that induced by B16F1 cell injection (∼66% of peak NO level of B16F1 cells, P < 0.01). The injection of saline alone was not associated with significantly increased NO production. Pretreatment of animals with the NO synthase inhibitor L-NAME inhibited NO production at 20 minutes by 64%. NO induction was completely absent in mice defective in the eNOS gene. NO was not detected in B16F1 cells (data not shown). To identify the cellular origin of NO, 2 × 106 fluorescent-labeled B16F1 cells were introduced into the perfusate of isolated intact WT mouse lungs in which the endothelium had been preloaded with DAF-2 DA. A low level of fluorescence, indicative of NO production, was seen after loading in all groups. Enhanced fluorescence was seen within 2 minutes of attachment to the pulmonary endothelium of WT mice. This fluorescence increased throughout the next 20 minutes at the site of arrest and also in adjacent endothelial cells. Fluorescence did not change from basal levels after arrest of the tumor cells in eNOS-deficient mice (Figure 2, Table 1).Table 1In Situ Quantification of NO Production by DAF-2 Fluorescence at Sites of B16F1 Melanoma Cell ArrestTime (minutes)WT C57Bl/6 mice (mean ± SE)eNOS KO mice (mean ± SE)000545 ± 10*P < 0.05 as compared to eNOS KO mice at 5 minutes.13 ± 610151 ± 46†P < 0.01 as compared to eNOS KO mice at 10, 15, and 20 minutes.31 ± 1315292 ± 99†P < 0.01 as compared to eNOS KO mice at 10, 15, and 20 minutes.33 ± 1320347 ± 119†P < 0.01 as compared to eNOS KO mice at 10, 15, and 20 minutes.43 ± 17Pulmonary endothelial cells were preloaded with DAF-2DA as described in Figure 2. Two ×106 B16F1 melanoma cells were added to the ex vivo perfusate and images were acquired. Endothelial cells adjacent to the arrested tumor cells were outlined and the fluorescence intensity was measured throughout time. For each lung, we determined the mean fluorescence intensities of four to seven endothelial cells and for each condition, three to four lungs were averaged. Data represent the percent increase in DAF-2T fluorescence intensity above baseline values and expressed as relative fluorescence intensities (arbitrary units). All results are expressed as the mean ± S.E. for each condition.* P < 0.05 as compared to eNOS KO mice at 5 minutes.† P < 0.01 as compared to eNOS KO mice at 10, 15, and 20 minutes. Open table in a new tab Pulmonary endothelial cells were preloaded with DAF-2DA as described in Figure 2. Two ×106 B16F1 melanoma cells were added to the ex vivo perfusate and images were acquired. Endothelial cells adjacent to the arrested tumor cells were outlined and the fluorescence intensity was measured throughout time. For each lung, we determined the mean fluorescence intensities of four to seven endothelial cells and for each condition, three to four lungs were averaged. Data represent the percent increase in DAF-2T fluorescence intensity above baseline values and expressed as relative fluorescence intensities (arbitrary units). All results are expressed as the mean ± S.E. for each condition. To determine whether there was a correlation between NO production and apoptosis, lung tissues were harvested at various times after injecting 5 × 105 fluoresbrite microsphere-labeled cells. Cryostat sections of the lung tissue were stained for apoptotic cells with the ApopTag kit. Tumor cells were identified by their fluorescence (Figure 3A) and scored for apoptosis (Figure 3B). Tumor cells became fragmented by 48 hours after injection (Figure 3C). Fragmentation was greater in WT mice than in eNOS KO mice or L-NAME-treated mice (Table 2). As shown in Figure 4A, apoptosis of tumor cells increased with time after injection in WT mice. Enhanced apoptosis could be seen as soon as 4 hours after cell injection, reached a peak value threefold greater than baseline at 8 hours, and remained detectable throughout a 48-hour period. Apoptosis of tumor cells did not increase above baseline values in mice defective in the eNOS gene or in mice pretreated with L-NAME.Table 2Tumour Cell Fragmentation after Arrest in the Pulmonary Microvasculature% Cells that are fragmented (mean ± SEM)Group8 hours48 hoursWT mice26 ± 256 ± 2WT mice + L-NAME22 ± 135 ± 2*P < 0.001 when compared" @default.
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• W2089986662 date "2003-02-01" @default.
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• W2089986662 title "Arrest of B16 Melanoma Cells in the Mouse Pulmonary Microcirculation Induces Endothelial Nitric Oxide Synthase-Dependent Nitric Oxide Release that Is Cytotoxic to the Tumor Cells" @default.
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• W2089986662 doi "https://doi.org/10.1016/s0002-9440(10)63835-7" @default.
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