Multimodal assessment of early tumor response to chemotherapy: comparison between diffusion-weighted MRI, 1H-MR spectroscopy of choline and USPIO particles targeted at cell death.

The aim of this study was to determine the value of different magnetic resonance (MR) protocols to assess early tumor response to chemotherapy. We used a murine tumor model (TLT) presenting different degrees of response to three different cytotoxic agents. As shown in survival curves, cyclophosphamide (CP) was the most efficient drug followed by 5-fluorouracil (5-FU), whereas the etoposide treatment had little impact on TLT tumors. Three different MR protocols were used at 9.4 Tesla 24 h post-treatment: diffusion-weighted (DW)-MRI, choline measurement by (1) H MRS, and contrast-enhanced MRI using ultrasmall iron oxide nanoparticles (USPIO) targeted at phosphatidylserine. Accumulation of contrast agent in apoptotic tumors was monitored by T(2) -weighted images and quantified by EPR spectroscopy. Necrosis and apoptosis were assessed by histology. Large variations were observed in the measurement of choline peak areas and could not be directly correlated to tumor response. Although the targeted USPIO particles were able to significantly differentiate between the efficiency of each cytotoxic agent and best correlated with survival endpoint, they present the main disadvantage of non-specific tumor accumulation, which could be problematic when transferring the method to the clinic. DW-MRI presents a better compromise by combining longitudinal studies with a high dynamic range; however, DW-MRI was unable to show any significant effect for 5-FU. This study illustrates the need for multimodal imaging in assessing tumor response to treatment to compensate for individual limitations.

Membrane-bound carbonic anhydrase in human retinal pigment epithelium.

PURPOSE: Inhibition of carbonic anhydrase (CA) by acetazolamide causes a decrease in the standing potential of the retinal pigment epithelium (RPE) and an increase in the rate of subretinal fluid absorption, and it may improve cystoid macular edema. These effects are thought to be mediated by the RPE. Given the solubility coefficient of acetazolamide, the drug is most likely to act by direct inhibition of membrane-bound CA (CA IV). To identify a substrate for acetazolamide in the RPE, the distribution of CA activity and the isoform of CA in the RPE membrane were investigated. METHODS: Carbonic anhydrase activity was determined by Hansson's technique in fresh human eyes from donors of both sexes and different ages. The presence of the membrane-bound isoform CA IV was investigated immunohistochemically at the light and electron microscopic level, as well as by Western blotting in fresh RPE, and in adult and fetal RPE cultures. RESULTS: Hansson's histochemical method demonstrated CA activity on the apical and basolateral cell membrane of the RPE. Using the gamma-globulin fraction of a polyclonal antibody against pure CA IV, immunocytochemistry showed labeling for CA IV on the apical RPE membrane or morphologically polarized human adult and fetal RPE cultures. Gel electrophoresis and Western blotting demonstrated a major immunoreactive band at 55 kDa in homogenates, which was consistently reduced to approximately 35 kDa by incorporation of 0.1% Triton X-100 detergent. CONCLUSIONS: These results suggest that the clinical effects of carbonic anhydrase inhibitors on RPE function may be mediated via membrane-bound carbonic anhydrase activity in RPE and that CA IV is responsible for activity on the apical surface.

Electron paramagnetic resonance as a sensitive tool to assess the iron oxide content in cells for MRI cell labeling studies.

MRI cell tracking is a promising technique to track various cell types (stem cells, tumor cells, etc.) in living animals. Usually, cells are incubated with iron oxides (T(2) contrast agent) in order to take up the particles before being injected in vivo. Iron oxide quantification is important in such studies for validating the labeling protocols and assessing the dilution of the particles with cell proliferation. We here propose to implement electron paramagnetic resonance (EPR) as a very sensitive method to quantify iron oxide concentration in cells. Iron oxide particles exhibit a unique EPR spectrum, which directly reflects the number of particles in a sample. In order to compare EPR with existing methods (Perls's Prussian blue reaction, ICP-MS and fluorimetry), we labeled tumor cells (melanoma and renal adenocarcinoma cell lines) and fibroblasts with fluorescent iron oxide particles, and determined the limits of detection of the different techniques. We show that EPR is a very sensitive technique and is specific for iron oxide quantification as measurements are not affected by endogenous iron. As a consequence, EPR is well adapted to perform ex vivo analysis of tissues after cell tracking experiments in order to confirm MRI results.

Iron oxide particles covered with hexapeptides targeted at phosphatidylserine as MR biomarkers of tumor cell death.

The aim of the study was to evaluate the ability of a new MR contrast agent to detect cell death as a biomarker of the efficacy of anti-cancer treatment. The phosphatidylserine-targeted hexapeptide (E3) was coupled to pegylated ultrasmall iron oxide nanoparticles (USPIO) that can be detected by magnetic resonance imaging (MRI) and by electron paramagnetic resonance (EPR). USPIO binding to staurosporine-treated TLT (transplantable liver tumor) cells, evaluated by X-Band EPR, indicated twice as much binding of USPIO grafted with the E3 peptide, compared with USPIO grafted with a scrambled peptide or ungrafted USPIO. In vivo experiments were carried out using TLT cells implanted intramuscularly into NMRI mice, and tumor cell death was induced by irradiation. After intravenous injection of the different types of USPIO, the accumulation of contrast agent was evaluated ex vivo by X-band EPR, in vivo by L-band EPR and by T(2)-weighted MRI. In irradiated tumors there was greater accumulation of the targeted USPIO particles compared with control particles or compared with the targeted particles in untreated tissues. In conclusion, phosphatidylserine-targeting of USPIO particles can detect dying tissues. This molecular targeted system should be evaluated further as a potential biomarker of tumor response to treatment.

Iron oxide based MR contrast agents: from chemistry to cell labeling.

Superparamagnetic iron oxide nanoparticles can be used for numerous applications such as MRI contrast enhancement, hyperthermia, detoxification of biological fluids, drug delivery, or cell separation. In this work, we will summarize the chemical routes for synthesis of iron oxide nanoparticles, the fluid stabilization, and the surface modification of superparamagnetic iron oxide nanoparticles. Some examples of the numerous applications of these particles in the biomedical field mainly as MRI negative contrast agents for tissue-specific imaging, cellular labeling, and molecular imaging will be given. Larger particles or particles displaying a non-neutral surface (thanks to their coating or to a cell transfection agent with which they are mixed) are very useful tools, although the cells to be labeled have no professional phagocytic function. Labeled cells can then be transplanted and monitored by MRI in a broad spectrum of applications. Direct in vivo magnetic labeling of cells is mainly performed by intravenous injection of long-circulating iron oxide-based MRI contrast agents, which can extravasate and/or undergo a cellular uptake in an amount sufficient to allow an MRI visualization of areas of interest such as inflamed regions or tumors. Particles with long circulation times, or able to induce a strong negative effect individually have been also modified by conjugation to a ligand, so that their cellular uptake, or at least their binding to the cell surface, could occur through a specific ligand-receptor interaction, in vivo as well as in vitro. Thus, experimentally as well as in a few trials on humans, iron oxide particles currently find promising applications.

Anion channels in a human pancreatic cancer cell line (Capan-1) of ductal origin.

Transepithelial solute transport and bicarbonate secretion are major functions of pancreatic duct cells, and both functions are thought to involve the presence of chloride channels in the apical membrane of the cell. After being isolated from a human pancreatic adenocarcinoma, the Capan-1 cell line conserves most of the properties of ductal cells and thus constitutes a useful system for investigating the role of chloride channels. Using patch-clamp techniques, we identified three different chloride-selective channels in the apical membrane of confluent Capan-1 cells. Two were non-rectifying chloride channels with low (50 pS) and high (350 pS) unitary conductances. Both channels were active in cell-attached recordings, and they were consistently located together in the same patch. Maxi Cl- channels displayed multiple subconductance states, and were reversibly inactivated by either positive or negative voltage changes, which indicates that they were optimally opened at the cell resting potential. The third was an outwardly rectifying chloride channel with a unitary conductance of 38 pS and 70 pS at negative and positive potentials respectively. Rectifying Cl- channels were clustered in discrete loci. They were silent in situ, but became active after patch excision. In inside-out excised patches, the three channels displayed a high selectivity for Cl- over monovalent cations (Na+ and K+) and gluconate. They were blocked by 20-200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and were insensitive to changes in the Ca2+ concentration. Our results show that the apical membrane of Capan-1 cells contains a high density of chloride channels; these channels may provide pathways for transepithelial solute transport as well as for bicarbonate secretion.

The expression of carbonic anhydrases II and IV in the human pancreatic cancer cell line (Capan 1) is associated with bicarbonate ion channels.

Human pancreatic ductal cells of the Capan 1 cell line differentiate progressively during growth. After the exponential growth phase, the cells elongate and become polarized with their apical poles covered by microvilli and separated from the basolateral pole by tight junctions. In this stationary phase, they form domes, which are thought to result from the exchange of water and electrolytes. In this study, we demonstrated, using patch-clamp techniques, that HCO3- ions exit via the g350 high conductance anionic channel we observed recently at the Capan 1 cell surface. This g350 channel was thought to be either a Cl-/HCO3- antiport or a simple HCO3- channel. The stilbene derivatives 4-acetamido-4 isothiocyano-2-2'-disulfonic acid (SITS) and 4,4' diisothiocyano stilbene-2,2' disulfonic acid (DIDS) reduced both the number of domes and the Cl- and HCO3- flux through the g350 channel. Moreover, using histochemical, immunocytochemical and biochemical methods we showed that Capan 1 cells express a specific pattern of carbonic anhydrases (CA). Two types of CA were detected: the CA II isozyme mainly localized in the cytoplasm, but also found beneath the inner leaflet of the apical plasma membrane, and the CA IV isozyme localized on the outer leaflet of the apical plasma membrane and microvilli. Their molecular masses were 30 (CA II) and 55 kDa (CA IV), respectively. They were expressed continuously during the exponential growth phase, although their activity increased greatly during the stationary phase. Inhibition of dome formation by acetazolamide indicated the existence of a direct relationship between dome formation and CA. Characteristic structures with a central electron-dense core surrounded by a light halo were observed on the surface of cell membrane using histochemical and immunocytochemical methods. These structures were thought to represent a channel, corresponding possibly to CA IV. Our observations suggest that Capan 1 cells, despite their neoplasic transformation, produce HCO3- ions in the same way as normal human pancreatic ductal cells. Capan 1 cells in culture may therefore represent a suitable model for studying pancreatic duct HCO3- secretion at the cellular and molecular levels.

[Membrane-bound carbonic anhydrase (CA IV) in human corneal epithelium and endothelium]. / Membrangebundene Carboanhydrase (CA IV) im menschlichen kornealen Epi- und Endothel.

PURPOSE: Active HCO3- transport through the corneal endothelial cell layer causes a dehydration of the corneal stroma and is thought to be driven by Na/K- and HCO3(-)-dependent ATPase as well as an electro-genic Na/HCO3- cotransport. Transmembrane bicarbonate transport has also been associated with the recently characterised membrane-anchored isoform of carbonic anhydrase (CA IV) in various tissues. We investigated the localisation of CA IV in human fresh and cultured epi- and endothelium at the light- (LM) and electron-microscopic (EM) level. METHODS: Postmortem corneas were obtained within 12 hours of death, stored in formaldehyde and sectioned in paraffin. LM immunohisto-chemistry was performed using the purified gamma-globulin fraction of a polyclonal chicken antibody against CA IV isolated from human kidneys. Epi- and endothelial cell cultures were grown in uncoated flasks under standard conditions and processed both for LM and EM immunohistochemistry using the same antibody. RESULTS: Lightmicroscopy of fresh tissue showed membrane staining for CA IV in the whole circumference of the endothelium. Little staining was also observed in some cells of the basal cell layer of the epithelium. Immunohistochemical staining at the EM level was confined to the cell surface of confluent cultures of both epi- and endothelial cells. CONCLUSION: The localisation of CA IV to the cell surface of fresh and cultured corneal endothelium suggests the presence of a membrane-bound ion exchange mechanism which may be important for HCO3- transport and corneal hydration. Compromising this mechanism by treatment with local carbonicanhydrase inhibitors may be of clinical importance in selected endothelial disease.

Carbonic anhydrase IV activity is localized on the exterior surface of human erythrocytes.

Carbonic anhydrase (CA) cytoplasmic isozymes CA I and CA II were found in human erythrocyte membrane ghosts, when prepared at pH 5.4 and pH 7.4, but not in ghosts prepared at pH 8.2. These findings could indicate that previously reported CA activity of ghosts was owing to contamination by CA I and CA II during the preparation of the ghosts. However, using a sensitive micro-assay, CA activity was also found in ghosts prepared at pH 8.2. This activity constitutes 0.2% of the erythrocytes' total CA-activity, and originates from a membrane-associated isoform of CA, located at the exterior membrane surface. It has immunochemical and kinetic properties like those of the membrane-bound CA IV, previously isolated from kidney, lung and small blood vessels. Its function is possibly to interact with the red cell membrane anionic transport protein, band 3, for the bicarbonate/chloride exchange.