ABSTRACT
Atypical chemokine receptor CXCR7 (ACKR3) functions as a scavenger receptor for chemokine CXCL12, a molecule that promotes multiple steps in tumor growth and metastasis in breast cancer and multiple other malignancies. Although normal vascular endothelium expresses low levels of CXCR7, marked upregulation of CXCR7 occurs in tumor vasculature in breast cancer and other tumors. To investigate effects of endothelial CXCR7 in breast cancer, we conditionally deleted this receptor from vascular endothelium of adult mice, generating CXCR7(ΔEND/ΔEND) animals. CXCR7(ΔEND/ΔEND) mice appeared phenotypically normal, although these animals exhibited a modest 35±3% increase in plasma CXCL12 as compared with control. Using two different syngeneic, orthotopic tumor implant models of breast cancer, we discovered that CXCR7(ΔEND/ΔEND) mice had significantly greater local recurrence of cancer following resection, elevated numbers of circulating tumor cells and more spontaneous metastases. CXCR7(ΔEND/ΔEND) mice also showed greater experimental metastases following intracardiac injection of cancer cells. These results establish that endothelial CXCR7 limits breast cancer metastasis at multiple steps in the metastatic cascade, advancing understanding of CXCL12 pathways in tumor environments and informing ongoing drug development targeting CXCR7 in cancer.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Endothelium, Vascular/metabolism , Receptors, CXCR/physiology , Animals , Cell Line, Tumor , Female , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neoplasm Metastasis , Receptors, CXCR/genetics , Receptors, CXCR/metabolism , Tumor Microenvironment/geneticsABSTRACT
Genetically encoded far-red and near-infrared fluorescent proteins enable efficient imaging in studies of tumorigenesis, embryogenesis, and inflammation in model animals. Here we report comparative testing of available GFP-like far-red fluorescent proteins along with a modified protein, named Katushka2S, and near-infrared bacterial phytochrome-based markers. We compare fluorescence signal and signal-to-noise ratio at various excitation wavelength and emission filter combinations using transiently transfected cell implants in mice, providing a basis for rational choice of optimal marker(s) for in vivo imaging studies. We demonstrate that the signals of various far-red fluorescent proteins can be spectrally unmixed based on different signal-to-noise ratios in different channels, providing the straightforward possibility of multiplexed imaging with standard equipment. Katushka2S produced the brightest and fastest maturing fluorescence in all experimental setups. At the same time, signal-to-noise ratios for Katushka2S and near-infrared bacterial phytochrome, iRFP720 were comparable in their optimal channels. Distinct spectral and genetic characteristics suggest this pair of a far-red and a near-infrared fluorescent protein as an optimal combination for dual color, whole body imaging studies in model animals.
Subject(s)
Luminescent Proteins/metabolism , Whole Body Imaging , Alternative Splicing , Animals , HEK293 Cells , Heterografts , Humans , Luminescent Proteins/genetics , Mice , Models, Animal , Molecular Imaging/methods , RNA Splice Sites , Signal-To-Noise Ratio , Whole Body Imaging/methods , Red Fluorescent ProteinABSTRACT
Compelling evidence shows that chemokine C-X-C motif chemokine ligand 12 (CXCL12) drives metastasis in multiple malignancies. Similar to other key cytokines in cancer, CXCL12 exists as several isoforms with distinct biophysical properties that may alter signaling and functional outputs. However, effects of CXCL12 isoforms in cancer remain unknown. CXCL12-α, -ß and -γ showed cell-type-specific differences in activating signaling through G protein-dependent pathways in cell-based assays, while CXCL12-γ had greatest effects on recruitment of the adapter protein ß-arrestin 2. CXCL12-ß and -γ also stimulated endothelial tube formation to a greater extent than CXCL12-α. To investigate the effects of CXCL12 isoforms on tumor growth and metastasis, we used a mouse xenograft model of metastatic human breast cancer combining CXCR4+ breast cancer cells and mammary fibroblasts secreting an isoform of CXCL12. Altough all CXCL12 isoforms produced comparable growth of mammary tumors, CXCL12-γ significantly increased metastasis to bone marrow and other sites. Breast cancer cells originating from tumors with CXCL12-γ fibroblasts upregulated RANKL (receptor activator of nuclear factor-κB ligand), contributing to bone marrow tropism of metastatic cancer cells. CXCL12-γ was expressed in metastatic tissues in mice, and we also detected CXCL12-γ in malignant pleural effusions from patients with breast cancer. In our mouse model, mammary fibroblasts disseminated to sites of breast cancer metastases, providing another mechanism to increase levels of CXCL12 in metastatic environments. These studies identify CXCL12-γ as a potent pro-metastatic molecule with important implications for cancer biology and effective therapeutic targeting of CXCL12 pathways.
Subject(s)
Bone Neoplasms/secondary , Breast Neoplasms/pathology , Chemokine CXCL12/metabolism , Animals , Arrestins/metabolism , Bone Neoplasms/genetics , Cells, Cultured , Chemokine CXCL12/genetics , Chemokine CXCL12/pharmacology , Female , Gene Expression Regulation, Neoplastic , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/pathology , Humans , Mice , Protein Isoforms/genetics , Protein Isoforms/metabolism , RANK Ligand/biosynthesis , Receptors, CXCR4/metabolism , Xenograft Model Antitumor Assays , beta-Arrestin 2 , beta-ArrestinsABSTRACT
Chemokines critically regulate chemotaxis in normal and pathologic states, but there is limited understanding of how multicellular interactions generate gradients needed for cell migration. Previous studies of chemotaxis of CXCR4+ cells toward chemokine CXCL12 suggest the requirement of cells expressing scavenger receptor CXCR7 in a source-sink system. We leveraged an established microfluidic device to discover that chemotaxis of CXCR4 cells toward distinct isoforms of CXCL12 required CXCR7 scavenging only under conditions with higher than optimal levels of CXCL12. Chemotaxis toward CXCL12-ß and -γ isoforms, which have greater binding to extracellular molecules and have been largely overlooked, was less dependent on CXCR7 than the more commonly studied CXCL12-α. Chemotaxis of CXCR4+ cells toward even low levels of CXCL12-γ and CXCL12-ß still occurred during treatment with a FDA-approved inhibitor of CXCR4. We also detected CXCL12-γ only in breast cancers from patients with advanced disease. Physiological gradient formation within the device facilitated interrogation of key differences in chemotaxis among CXCL12 isoforms and suggests CXCL12-γ as a biomarker for metastatic cancer.
Subject(s)
Breast Neoplasms/immunology , Chemokine CXCL12/immunology , Chemotaxis/immunology , Receptors, CXCR/immunology , Animals , Benzylamines , Cell Line, Tumor , Chemokine CXCL12/genetics , Cyclams , Female , Heterocyclic Compounds/pharmacology , Humans , Mice, Inbred C57BL , Microfluidics , Microscopy, Fluorescence , Microscopy, Phase-Contrast , Protein Isoforms/genetics , Protein Isoforms/immunology , RNA, Neoplasm/chemistry , RNA, Neoplasm/genetics , Receptors, CXCR/antagonists & inhibitors , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Chemokine CXCL12 and receptor CXCR4 control multiple steps in primary tumor growth and metastasis in breast cancer and more than 20 other human malignancies. Mechanisms that regulate availability of CXCL12 in tumor microenvironments will substantially impact cancer progression and ongoing efforts to target the CXCL12-CXCR4 pathway for cancer chemotherapy. We used dual luciferase imaging to investigate CXCR7-dependent scavenging of CXCL12 in breast tumors in vivo and quantify effects of CXCR7 on tumor growth and metastasis of a separate population of CXCR4+ breast cancer cells. In a mouse xenograft model of human breast cancer, in vivo imaging showed that malignant cells expressing CXCR7 reduced bioluminescent CXCL12 secreted in the primary tumor microenvironment. Capitalizing on sensitive detection of bioluminescent CXCL12, we also demonstrated that CXCR7+ cells reduced amounts of chemokine released from orthotopic tumors into the circulation. Immunofluorescence staining of human primary breast cancers showed expression of CXCR4 and CXCR7 on malignant cells in ≈30% of cases. In most cases, CXCR4 and CXCR7 predominantly were expressed on separate populations of malignant cells in a tumor. We modeled these cases of human breast cancer by co-implanting tumor xenografts with CXCR4+ breast cancer cells, human mammary fibroblasts secreting CXCL12, and CXCR7+ or control breast cancer cells. Bioluminescence imaging showed that CXCR7+ breast cancer cells enhanced proliferation of CXCR4+ breast cancer cells in orthotopic tumors and spontaneous metastases. Treatment with a small-molecule inhibitor of CXCR7 chemokine limited the growth of CXCR4+ breast cancer cells in tumors that also contained malignant CXCR7+ cells. These studies establish a new in vivo imaging method to quantify chemokine scavenging by CXCR7 in the tumor microenvironment and identify that CXCR7+ cells promote growth and metastasis of CXCR4+ breast cancer cells.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Chemokine CXCL12/metabolism , Receptors, CXCR4/metabolism , Receptors, CXCR/metabolism , Animals , Breast Neoplasms/genetics , Cell Line, Transformed , Cell Membrane/metabolism , Cell Proliferation , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Neoplasm Metastasis , Receptors, CXCR/antagonists & inhibitors , Receptors, CXCR/genetics , Receptors, CXCR4/genetics , Tumor Burden , Tumor Microenvironment/geneticsABSTRACT
CXCR7 is a receptor for chemokines including CXCL12 (stromal-derived factor-1), a molecule that promotes tumor growth and metastasis in breast cancer and other malignancies. Building on the recent observation that CXCR7 sequesters CXCL12, we investigated mechanisms for CXCR7-dependent uptake of chemokines. Breast cancer cells expressing CXCR7 accumulated chemokines CXCL12 and CXC11 present at concentrations <1 ng/ml, unlike cells expressing CXCR4. CXCR7-dependent accumulation of chemokines was reduced by inhibitors of clathrin-mediated endocytosis. After CXCR7-mediated internalization, CXCL12 trafficked to lysosomes and was degraded, although levels of CXCR7 remained stable. CXCR7 reduced CXCL12 in the extracellular space, limiting the amounts of chemokine available to acutely stimulate signaling through CXCR4. CXCR7 constitutively internalized and recycled to the cell membrane even in the absence of ligand, and addition of chemokines did not significantly enhance receptor internalization. Chemokines at concentrations less than the Kd values for ligand-receptor binding did not alter levels of CXCR7 at the cell surface. Higher concentrations of chemokine ligands reduced the total cell surface expression of CXCR7 without affecting receptor internalization, indicating that receptor recycling was inhibited. CXCR7-dependent uptake of chemokines and receptor trafficking were regulated by beta-arrestin 2. These studies establish mechanisms through which CXCR7 regulates the availability of chemokine ligands in the extracellular space.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Chemokines/metabolism , Receptors, CXCR/metabolism , Arrestins/metabolism , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Membrane/metabolism , Chemokine CXCL11/metabolism , Chemokine CXCL12/metabolism , Clathrin/metabolism , Cytosol/metabolism , Endocytosis , Extracellular Space/metabolism , Gene Expression Regulation, Neoplastic , Humans , Ligands , Lysosomes/metabolism , Protein Transport , Receptors, CXCR4/metabolism , beta-Arrestin 2 , beta-ArrestinsABSTRACT
Microscale biopatterning enables regulation of cell-material interactions and cell shape, and enables multiplexed high-throughput studies in a cell- and reagent-efficient manner. The majority of available techniques rely on physical contact of a stamp, pin, or mask with mainly a dry surface. Inkjet and piezoelectric printing is carried out in a non-contact manner but still requires a substantially dry substrate to ensure fidelity of printed patterns. These existing methods, therefore, are limited for patterning onto delicate surfaces of living cells because physical contact or substantially dry conditions are damaging to them. Microfluidic patterning with laminar streams does enable non-contact patterning in fully aqueous environments but with limited throughput and reagent diffusion across interfacial flows. Here, we describe a polymeric aqueous two-phase system that enables patterning nanolitres of a reagent-containing aqueous phase, in arbitrary shapes, within a second aqueous phase covering a cell monolayer. With the appropriate medium formulation, reagents of interest remain confined to the patterned phase without significant diffusion. The fully aqueous environment ensures high reagent activity and cell viability. The utility of this strategy is demonstrated with patterned delivery of genetic materials to mammalian cells for phenotypic screening of gene expression and gene silencing.
Subject(s)
Cells/metabolism , Drug Delivery Systems , Gene Expression Profiling/methods , Gene Silencing , Water/chemistry , Animals , Biological Transport , Cell Line , Cell Survival , Cells/cytology , Humans , Indicators and Reagents/metabolism , Microchemistry , Oligonucleotide Array Sequence Analysis , PhenotypeABSTRACT
MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Biological Transport/physiology , Drug Resistance, Multiple/physiology , Membrane Potentials/physiology , Adenocarcinoma , Breast Neoplasms , Cell Membrane/metabolism , Female , Humans , Ionophores/pharmacology , Organotechnetium Compounds/metabolism , Patch-Clamp Techniques , Tumor Cells, Cultured , Valinomycin/pharmacologyABSTRACT
Class I P-glycoproteins [Pgp; MDR1 (ABCB1) in humans, mdr1a and mdr1b in mice] confer resistance to structurally diverse chemotherapeutic drugs in cultured cells and intact animals, but the function of these proteins in normal physiology remains poorly characterized. Based on studies in cell culture, a putative role for class I Pgp in absorption and intracellular trafficking of sterols has been proposed. We examined wild-type and mdr1a(-/)-/1b(-/)- mice to determine whether class I Pgp affects cholesterol absorption and esterification in vivo. Using a dual-isotope protocol, absorption of orally administered radiolabeled cholesterol into serum did not differ between wild-type and mdr1a(-/)-/1b(-/)- mice, demonstrating that class I Pgp is not essential for overall absorption of cholesterol through the intestine. However, the ratio of oral to intravenous labeled cholesterol in liver was decreased significantly in mdr1a(-/)-/1b(-/)- mice. In the liver, but not other tested organs, deletion of class I Pgp enhanced kinetics of esterification of an oral bolus of radiolabeled cholesterol without affecting esterification of cholesterol administered intravenously. Steady-state hepatic content of cholesterol and esterified cholesterol also were unaffected by absence of mdr1a and mdr1b.Thus, in normal animals, class I Pgp functions to kinetically increase hepatic accumulation and decrease esterification of orally administered cholesterol in vivo.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/deficiency , Cholesterol Esters/metabolism , Cholesterol/pharmacokinetics , Liver/metabolism , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/physiology , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/physiology , Absorption , Animals , Carbon Radioisotopes , Cholesterol/administration & dosage , Cholesterol/metabolism , Cholesterol Esters/analysis , Cholesterol, Dietary/administration & dosage , Cholesterol, Dietary/pharmacokinetics , Esterification , Injections, Intravenous , Kinetics , Liver/chemistry , Male , Mice , Tritium , ATP-Binding Cassette Sub-Family B Member 4ABSTRACT
Rapid and efficient delivery of radioactive metal complexes to the cell interior would enable novel applications in medical imaging and radiotherapy. Membrane permeant peptide conjugates incorporating HIV-1 Tat transactivation protein sequences (GRKKRRQRRR) and an appropriate peptide-based motif (epsilon-KGC) that provides an N(3)S donor core for chelating technetium and rhenium were synthesized. Oxotechnetium(V) and oxorhenium(V) Tat-peptide complexes were prepared by facile transchelation reactions with permetalates, tin(II) chloride and sodium glucoheptonate. RP-HPLC showed two major [(99m)Tc]Tat-peptide species (4) that differed in retention time by approximately 2 min corresponding to two [Re]Tat-peptide species (7) shown to have identical mass, consistent with formation of two isomers, likely the oxo-metal diastereomers. [(99m)Tc]Tat-peptides were stable to transchelation in vitro. In human Jurkat cells, [(99m)Tc]Tat-peptide 4 showed concentrative cell accumulation (30-fold greater than extracellular concentration) and rapid uptake kinetics (t(1/2) < 2 min) in a diastereomeric-comparable manner. Paradoxically, uptake was enhanced in 4 degrees C buffer compared to 37 degrees C, while depolarization of membrane potential as well as inhibition of microtubule function and vesicular trafficking showed no inhibitory effect. Cells preloaded with 4 showed rapid washout kinetics into peptide-free solution. Modification of [(99m)Tc]Tat-peptide by deletion of the N-terminus Gly with or without biotinylation minimally impacted net cell uptake. In addition, the C-terminus thiol of the prototypic Tat-peptide was labeled with fluorescein-5-maleimide to yield conjugate 8. Fluorescence microscopy directly localized conjugate 8 to the cytosol and nuclei (possibly nucleolus) of human Jurkat, KB 3-1 and KB 8-5 tumor cells. Preliminary imaging studies in mice following intravenous administration of prototypic [(99m)Tc]Tat-peptide 4 showed an initial whole body distribution and rapid clearance by both renal and hepatobiliary excretion. Analysis of murine blood in vivo and human serum ex vivo revealed >95% intact complex, while murine urine in vivo showed 65% parent complex. Thus, these novel Tat-peptide chelate conjugates, capable of forming stable [Tc/Re(V)]complexes, rapidly translocate across cell membranes into intracellular compartments and can be readily derivatized for further targeted applications in molecular imaging and radiotherapy.
Subject(s)
Chelating Agents/metabolism , Gene Products, tat/chemistry , Organotechnetium Compounds/metabolism , Peptide Fragments/metabolism , Radiopharmaceuticals/metabolism , Radiotherapy , Rhenium/metabolism , Amino Acid Sequence , Animals , Chelating Agents/chemistry , HIV-1/metabolism , Humans , Mice , Mice, Inbred BALB C , Organotechnetium Compounds/pharmacokinetics , Organotechnetium Compounds/therapeutic use , Peptide Fragments/chemistry , Peptide Fragments/pharmacokinetics , Radiopharmaceuticals/therapeutic use , Rhenium/pharmacokinetics , Rhenium/therapeutic use , Tissue Distribution , tat Gene Products, Human Immunodeficiency VirusABSTRACT
Multidrug resistance P-glycoprotein (Pgp) has been reported to localize in low-density, cholesterol-enriched membranes. However, effects of low-density membrane domains on function of Pgp remain unexplored in whole cell systems. In cells that express modest levels of the protein endogenously or through drug selection, Pgp predominantly localized to low-density membranes following separation on a sucrose gradient. When highly overexpressed in NIH 3T3 cells, a prominent amount of Pgp also was detected in high-density membranes. Removing cholesterol from cells with beta-methylcyclodextrin (CD), a sterol acceptor molecule, shifted fractions that contained Pgp from low toward high density, and this effect was reversed to a similar extent by restoring sterols with either cholesterol or enantiomeric cholesterol. However, function of human MDR1 Pgp as probed with Tc-Sestamibi, a transport substrate for Pgp, was not dependent on localization of Pgp in cholesterol-enriched membranes. Specific inhibition of MDR1 Pgp with GF120918 or LY335979 also was independent of cholesterol. Cell-type-specific effects of cholesterol content on function of human Pgp were detected by use of daunomycin, another substrate for Pgp, although efficacy of inhibitors remained independent of cholesterol. Conversely, both function and inhibition of hamster Pgp as measured with Tc-Sestamibi and daunomycin were in part dependent on normal cell content of cholesterol. These data show that Pgp preferentially localizes to low-density, cholesterol-enriched membrane domains, but acute depletion of cholesterol impacts Pgp-mediated drug transport in a substrate- and cell-type-specific manner.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cholesterol, LDL/metabolism , Cholesterol/pharmacology , 3T3 Cells , ATP Binding Cassette Transporter, Subfamily B, Member 1/drug effects , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Cells, Cultured , Cholesterol/chemistry , Cholesterol, LDL/chemistry , Cholesterol, LDL/drug effects , Daunorubicin/metabolism , Humans , Mice , Microscopy, Fluorescence , Stereoisomerism , Technetium Tc 99m Sestamibi/metabolismABSTRACT
Multidrug resistance (MDR1) P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP1), and breast cancer resistance protein (BCRP/MXR/ABCP) are members of the ATP-binding-cassette (ABC) superfamily of membrane transporters and are thought to function as energy-dependent efflux pumps of a variety of structurally diverse chemotherapeutic agents. We herein report the characterization of (99m)Tc-Tetrofosmin, a candidate radiopharmaceutical substrate of ABC transporters. (99m)Tc-Tetrofosmin showed high membrane potential-dependent accumulation in drug-sensitive KB 3-1 cells and low antagonist-reversible accumulation in MDR KB 8-5 and KB 8-5-11 cells in proportion to levels of MDR1 Pgp expression. In KB 8-5 cells, EC(50) values of the potent MDR antagonists N-(4-[2-(1,2,3, 4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9, 10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), (2R)-anti-5-¿3-[4-(10, 11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2 -hydroxypropoxy ¿quinoline trihydrochloride (LY335979), and (3'-keto-Bmt')-[Val(2)]-cyclosporin A (PSC 833) were 40, 66, and 986 nM, respectively. Furthermore, only baculoviruses carrying human MDR1, but not MDR3, conferred both a decrease in accumulation of (99m)Tc-Tetrofosmin in host Spodoptera frugiperda (Sf9) cells and a GF120918-induced enhancement. Transport studies with a variety of stably transfected and drug-selected tumor cell lines were performed with (99m)Tc-Tetrofosmin and compared with (99m)Tc-Sestamibi, a previously validated MDR imaging agent. MDR1 Pgp readily transported each agent. To a lesser extent, MRP1 also transported each agent, likely as co-transport substrates with GSH; neither agent was a substrate for the BCRP/MXR/ABCP half-transporter. In mdr1a(-/-) and mdr1a/1b(-/-) mice, (99m)Tc-Tetrofosmin showed approximately 3. 5-fold greater brain uptake and retention compared with wild-type, with no net change in blood pharmacokinetics, consistent with transport in vivo by Pgp expressed at the capillary blood-brain barrier. Molecular imaging of the functional transport activity of ABC transporters in vivo with (99m)Tc-Tetrofosmin and related radiopharmaceuticals may enable non-invasive monitoring of chemotherapeutic and MDR gene therapy protocols.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Neoplasm Proteins , Organophosphorus Compounds/metabolism , Organotechnetium Compounds/metabolism , 3T3 Cells , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Animals , Baculoviridae/physiology , Biological Transport , Blood-Brain Barrier , Cross Reactions , Humans , KB Cells , Mice , Radiopharmaceuticals/metabolism , Reproducibility of Results , Subcellular Fractions , Tumor Cells, CulturedABSTRACT
OBJECTIVE: Our aim is to determine whether agreement occurs between soft-copy (at a review workstation) and hard-copy (on laser-printed film) interpretations of fluoroscopic voiding cystourethrograms in neonates, infants, and children. SUBJECTS AND METHODS: Voiding cystourethrography was performed on 74 children (range, 2 weeks to 11 years old; mean, 3 years 6 months) for the evaluation of vesicoureteral reflux. The right and left ureters were scored separately by two observers on a scale of 0-5 using the international grading standard. Differences were tested for statistical significance with a marginal homogeneity test, and the strength of agreement was assessed using the kappa statistic. RESULTS: Of the 148 ureters evaluated, 39 showed vesicoureteral reflux and 109 showed no vesicoureteral reflux on both soft copy and hard copy. For 128 of 148 evaluations, interpretations of soft copy and hard copy produced agreement as to the grade of vesicoureteral reflux. For 11 of the 20 ureters with divergent interpretations, hard copy was scored one grade lower than soft copy; for the remaining nine ureters, hard copy was scored one grade higher than soft copy. No score differed by more than one grade. We found no statistically significant difference between soft-copy and hard-copy scores (p = .65), and agreement using the kappa statistic was substantial (.68). CONCLUSION: Soft-copy interpretation of voiding cystourethrograms is similar to hard-copy interpretation for vesicoureteral reflux.
Subject(s)
Fluoroscopy , Vesico-Ureteral Reflux/diagnostic imaging , Child , Child, Preschool , Cohort Studies , Humans , Infant , Infant, Newborn , Observer Variation , Radiographic Image Enhancement , Radiology Information Systems , Urethra/diagnostic imaging , Urinary Bladder/diagnostic imaging , Urination , X-Ray FilmABSTRACT
Class I P-glycoproteins (Pgp) confer multidrug resistance in tumors, but the physiologic function of Pgp in normal tissues remains uncertain. In cells derived from tissues that normally express Pgp, recent data suggest a possible role for Pgp in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. We investigated the esterification of plasma membrane cholesterol under basal conditions and in response to sphingomyelinase treatment in transfected and drug-selected cell lines expressing differing amounts of functional class I Pgp. Compared with parental NIH 3T3 fibroblasts, cells transfected with human multidrug resistance (MDR1) Pgp esterified more cholesterol both without and with sphingomyelinase. Esterification also was greater in drug-selected Dox 6 myeloma cells than parental 8226 cells, which express low and non-immunodetectable amounts of Pgp, respectively. However, no differences in total plasma membrane cholesterol were detected. Transfection of fibroblasts with the multidrug resistance-associated protein (MRP) did not alter esterification, showing that cholesterol trafficking was not generally affected by ATP-binding cassette transporters. Steroidal (progesterone, dehydroepiandrosterone) and non-steroidal antagonists (verapamil, PSC 833, LY335979, and GF120918) were evaluated for effects on both cholesterol trafficking and the net content of 99mTc-Sestamibi, a reporter of drug transport activity mediated by Pgp. In Pgp-expressing cells treated with nonselective and selective inhibitors, both the kinetics and efficacy of inhibition of cholesterol esterification differed from the antagonism of drug transport mediated by Pgp. Thus, although the data show that greater expression of class I Pgp within a given cell type is associated with enhanced esterification of plasma membrane cholesterol in support of a physiologic function for Pgp in facilitating cholesterol trafficking, the molecular mechanism is dissociated from the conventional drug transport activity of Pgp.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cholesterol/metabolism , Tetrahydroisoquinolines , 3T3 Cells , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Acridines/pharmacology , Animals , Cell Membrane/metabolism , Cyclosporins/pharmacology , Dehydroepiandrosterone/pharmacology , Dibenzocycloheptenes/pharmacology , Drug Resistance, Multiple , Esterification , Humans , Isoquinolines/pharmacology , Mice , Progesterone/pharmacology , Quinolines/pharmacology , Stereoisomerism , Tumor Cells, Cultured , Verapamil/pharmacologyABSTRACT
OBJECTIVE: The objective of our study was to compare child abuse detection using screen-film radiographs and their digitized images displayed on a computer workstation. MATERIALS AND METHODS: Skeletal surveys of 20 consecutive child abuse patients whose abuse was clinically proven by a combination of history, physical and radiographic findings, and social work history, and 20 consecutive control subjects were evaluated. Three radiologists rated both the screen-film radiographs (400-speed, double-emulsion film) and their digitized images displayed on a workstation (2K x 2K resolution) using a six-point ordinal scale for suspicion of child abuse, fracture detection, and image quality. The rating response was analyzed using multiobserver-multicase receiver operating characteristic analysis of variance. The McNemar test was used to evaluate differences between imaging techniques and between diagnoses made using each imaging technique and clinically proven child abuse. RESULTS: The area under the receiver operating characteristic curve for screen-film radiographs was 0.934+/-0.025 and for digitized images was 0.922+/-0.013. This difference was not significant (p = .658); however, two observers significantly underestimated the child abuse diagnosis with digitized images (p = .02). In a review of the false-negative child abuse diagnoses, observers failed to recognize characteristic metaphyseal fractures (10 observations) and rib fractures (five observations) on digitized images that had been recognized on screen-film radiographs. Mean image quality was rated significantly lower (p < .0001) and interpretation time was significantly longer (75 sec; p < .001) for the digitized images than for screen-film radiographs. CONCLUSION: The characteristic types of fractures that were not identified on the digitized images, lower image quality, and longer interpretation time raise concern that digitized images may not be adequate for interpretation of suspected child abuse.
Subject(s)
Bone and Bones/diagnostic imaging , Child Abuse/diagnosis , Fractures, Bone/diagnostic imaging , Radiographic Image Enhancement , False Negative Reactions , Female , Humans , Infant , Observer Variation , ROC Curve , Sensitivity and Specificity , X-Ray Intensifying ScreensABSTRACT
UNLABELLED: Overexpression of the multidrug resistance (MDR1) P-glycoprotein (Pgp) correlates with cancer chemotherapeutic failure. Lipophilic cationic radiopharmaceuticals such as 99mTc-sestamibi, 99mTc-tetrofosmin and 99Tc-furifosmin (Tc-Q12) have been validated as transport substrates for the MDR1 Pgp and may enable functional imaging of the MDR phenotype in cancer by observing enhanced washout rates of the tracers in those tumor areas expressing Pgp. To further explore and optimize the Pgp recognition properties of Schiff base phosphine mixed-ligand complexes of the Tc-Q series of nonreducible (Tc(III) cations, a variety of Tc-Q complexes were synthesized and tested in vitro for recognition as transport substrates by the human MDR1 Pgp. METHODS: Tracer assays with human drug-sensitive KB-3-1 epidermal carcinoma and MDR KB-8-5 cells expressing nonimmunodetectable and modest levels of MDR1 Pgp, respectively, were used to screen and pharmacologically characterize 37 novel 99mTc-Q analogs. RESULTS: The ideal agent should have low nonspecific binding, high distinction in net uptake between drug-sensitive cells and MDR tumor cells, and high enhancement of uptake in resistant cells after treatment with an MDR modulator, indicating selective blockade of Pgp-mediated efflux of the radiotracer. Three analogs, trans-[5,5'-(1,2-ethanediyldiimino)bis(2-OEt-2-Me-4-penten-3 -one)]bis[dimethyl(3-OMe-1-propyl)phosphine]99mTc(III) (99mTc-Q63) and two trans-[bis(methyl-bis(3-OMe-1-propyl)phosphine)] analogs (99mTc-Q57 and 99mTc-Q58) displayed transport distinctions between drug-sensitive and MDR cell lines that were equal to or greater than all previously available agents. Cyclosporin A, an MDR modulator, had no significant effect in KB-3-1 cells for these 99mTc-complexes but enhanced tracer accumulations in KB-8-5 cells with IC50 values of approximately 1 microM. In contrast, the non-MDR agents methotrexate and cisplatin had no effect on accumulation of 99mTc-Q complexes and 99mTc-sestamibi in KB-8-5 cells. CONCLUSION: Technetium-99m-Q57, 99mTc-Q58 and 99mTc-Q63 are avid transport substrates recognized by the human MDR1 Pgp, and have enhanced in vitro properties that may enable functional imaging of Pgp in vivo with improved signal-to-noise ratios and tissue contrast compared to currently available agents.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1 , Drug Resistance, Neoplasm , Organotechnetium Compounds , Radiopharmaceuticals , Humans , Organophosphorus Compounds , Technetium Tc 99m Sestamibi , Tumor Cells, CulturedABSTRACT
The multidrug resistance (MDR1) P-glycoprotein functions as a broad specificity efflux transporter of structurally diverse natural product and xenobiotic compounds. P-glycoprotein also is an important component of the functional blood-brain barrier. To enable further studies of function and modulation of MDR1 P-glycoprotein in vitro and in vivo, two novel phosphine technetium(III) complexes were designed and characterized: trans-[2,2'-(1, 2-ethanediyldiimino)bis(1, 5-methoxy-5-methyl-4-oxo-hexenyl)]bis[methylbis(3-methoxy-1- propyl)ph osphine]Tc(III) (Tc-Q58) and trans-[5,5'-(1,2-ethanediyl diimino)bis(2-ethoxy-2-methyl-3-oxo-4-pentenyl)]bis[dimethyl(3- methox y-1-propyl)phosphine)]Tc(III) (Tc-Q63). In human drug-sensitive KB 3-1 cells and multidrug-resistant KB 8-5 and 8-5-11 derivative cell lines, expressing nonimmunodetectable, low, and high levels of MDR1 P-glycoprotein, respectively, accumulation of Tc-Q58 and Tc-Q63 was inverse to expression of the transporter. Differences between drug-sensitive and multidrug-resistant cells, while detectable at picomolar concentrations of each radiopharmaceutical, were independent of tracer concentration. Ratios of tracer accumulation in KB 3-1 and 8-5 cells were 62.3 and 48.1 for Tc-Q58 and Tc-Q63, respectively. Cell contents of Tc-Q58 and Tc-Q63 were enhanced up to 60-fold in MDR cells by known modulators of MDR1 P-glycoprotein, while drugs not in the multidrug-resistant phenotype had no effect on their accumulation. In KB 8-5 cells, potency of modulators was GF120918 >> cyclosporin A > verapamil. Accumulation of Tc-Q58 and Tc-Q63 in Sf9 insect cells infected with a recombinant baculovirus containing human MDR1 P-glycoprotein was reduced in a GF120918-reversible manner (EC50 = 70 nM) compared with cells infected with a wild-type baculovirus. By contrast, cell contents of Tc-Q58 or Tc-Q63 in Sf9 cells expressing the homologous MDR3 P-glycoprotein did not differ from wild-type virus. Demonstrating molecular targeting of these complexes in vivo, distribution and retention of Tc-Q58 in brain tissue of FVB mice treated with a saturating dose of GF120918 and mice deficient in the mdr1a gene [mdr1a (-/-)] were enhanced 180% and 520% over control, respectively. Exploiting the gamma-emission spectrum of 99mTc, increased uptake of Tc-Q58 in brain tissue of mdr1a (-/-) mice was readily detected noninvasively by scintigraphic imaging. Thus, both Tc-Q58 and Tc-Q63 are demonstrated to be substrates for transport by MDR1 P-glycoprotein, broadening the specificity of this transporter to include phosphine-containing metal complexes. As shown with Tc-Q58, these Q complexes can be used to detect transport activity and modulation of MDR1 P-glycoprotein in vitro and to directly monitor the functional status of P-glycoprotein at the blood-brain barrier in vivo.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B , ATP-Binding Cassette Transporters/metabolism , Blood-Brain Barrier/physiology , Molecular Probes , Organophosphorus Compounds/metabolism , Organotechnetium Compounds/metabolism , Tetrahydroisoquinolines , ATP Binding Cassette Transporter, Subfamily B, Member 1/drug effects , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP-Binding Cassette Transporters/drug effects , ATP-Binding Cassette Transporters/genetics , Acridines/pharmacology , Animals , Biological Transport , Brain/diagnostic imaging , Isoquinolines/pharmacology , Mice , Mice, Mutant Strains , Radionuclide Imaging/methods , Recombinant Proteins/metabolism , Tumor Cells, CulturedABSTRACT
UNLABELLED: Overexpression of the multidrug resistance (MDR1) P-glycoprotein (Pgp) has been documented in nearly all forms of human cancers and increased levels of Pgp in some tumors correlate with poor response to treatment. Technetium-99m-sestamibi has recently been validated as a Pgp transport substrate. Pgp is also normally expressed along the biliary canalicular surface of hepatocytes and the luminal side of proximal tubule cells in the kidney, while not expressed in heart. METHODS: Focused on these organs with known Pgp status, we present the findings on 99mTc-sestamibi scintigraphy of three patients with refractory cancer who were imaged before and after administration of SDZ PSC 833, a second-generation, high-potency modulator of Pgp. RESULTS: Before treatment with SDZ PSC 833, scintigraphy using 99mTc-sestamibi showed normal, prompt clearance of the radiotracer from the liver and kidneys relative to the heart. After administration of the Pgp modulator, 99mTc-sestamibi was selectively retained in the liver and kidneys. CONCLUSION: Hepatobiliary and renal clearance of 99mTc-sestamibi are Pgp-mediated, and inhibition of Pgp transport in these organs can be successfully imaged using 99mTc-sestamibi in patients. Similar results might be expected with this and related radiopharmaceuticals for functional imaging of Pgp transport and modulation in tumors.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Drug Resistance, Multiple , Kidney/metabolism , Liver/metabolism , Neoplasm Recurrence, Local/drug therapy , Technetium Tc 99m Sestamibi/pharmacokinetics , Adenocarcinoma/diagnostic imaging , Adenocarcinoma/drug therapy , Breast Neoplasms/diagnostic imaging , Breast Neoplasms/drug therapy , Carcinoma, Ductal, Breast/diagnostic imaging , Carcinoma, Ductal, Breast/drug therapy , Cyclosporins/administration & dosage , Cystadenocarcinoma/diagnostic imaging , Cystadenocarcinoma/drug therapy , Female , Humans , Kidney/diagnostic imaging , Liver/diagnostic imaging , Lung Neoplasms/diagnostic imaging , Lung Neoplasms/drug therapy , Middle Aged , Neoplasm Recurrence, Local/diagnostic imaging , Ovarian Neoplasms/diagnostic imaging , Ovarian Neoplasms/drug therapy , Paclitaxel/administration & dosage , Radionuclide ImagingABSTRACT
MR imaging in the evaluation of noncardiac chest diseases is used primarily to supplement CT examinations. Because of its high soft-tissue contrast and ability to detect flowing blood, MR imaging has significant advantages for assessing tissue characteristics and vascular pathologic conditions. This article reviews imaging techniques and the MR appearance of mediastinal vascular anomalies, mediastinal and chest wall tumors, and post-treatment changes.