Martin Christlieb

A comparison of the behavior of (64)Cu-acetate and (64)Cu-ATSM in vitro and in vivo.

64Cu-diacetyl-bis(N4-methylthiosemicarbazonate), 64Cu-ATSM, continues to be investigated clinically as a PET agent both for delineation of tumor hypoxia and as an effective indicator of patient prognosis, but there are still aspects of the mechanism of action that are not fully understood. Methods: The retention of radioactivity in tumors after administration of 64Cu-ATSM in vivo is substantially higher for tumors with a significant hypoxic fraction. This hypoxia-dependent retention is believed to involve the reduction of Cu-ATSM, followed by the loss of copper to cellular copper processing. To shed light on a possible role of copper metabolism in hypoxia targeting, we have compared 64Cu retention in vitro and in vivo in CaNT and EMT6 cells or cancers after the administration of 64Cu-ATSM or 64Cu-acetate. Results: In vivo in mice bearing CaNT or EMT6 tumors, biodistributions and dynamic PET data are broadly similar for 64Cu-ATSM and 64Cu-acetate. Copper retention in tumors at 15 min is higher after injection of 64Cu-acetate than 64Cu-ATSM, but similar values result at 2 and 16 h for both. Colocalization with hypoxia as measured by EF5 immunohistochemistry is evident for both at 16 h after administration but not at 15 min or 2 h. Interestingly, at 2 h tumor retention for 64Cu-acetate and 64Cu-ATSM, although not colocalizing with hypoxia, is reduced by similar amounts by increased tumor oxygenation due to inhalation of increased O2. In vitro, substantially less uptake is observed for 64Cu-acetate, although this uptake had some hypoxia selectivity. Although 64Cu-ATSM is stable in mouse serum alone, there is rapid disappearance of intact complex from the blood in vivo and comparable amounts of serum bound activity for both 64Cu-ATSM and 64Cu-acetate. Conclusion: That in vivo, in the EMT6 and CaNT tumors studied, the distribution of radiocopper from 64Cu-ATSM in tumors essentially mirrors that of 64Cu-acetate suggests that copper metabolism may also play a role in the mechanism of selectivity of Cu-ATSM.

Automatic segmentation of adherent biological cell boundaries and nuclei from brightfield microscopy images

The detection and segmentation of adherent eukaryotic cells from brightfield microscopy images represent challenging tasks in the image analysis field. This paper presents a free and open-source image analysis package which fully automates the tasks of cell detection, cell boundary segmentation, and nucleus segmentation in brightfield images. The package also performs image registration between brightfield and fluorescence images. The algorithms were evaluated on a variety of biological cell lines and compared against manual and fluorescence-based ground truths. When tested on HT1080 and HeLa cells, the cell detection step was able to correctly identify over 80% of cells, whilst the cell boundary segmentation step was able to segment over 75% of the cell body pixels, and the nucleus segmentation step was able to correctly identify nuclei in over 75% of the cells. The algorithms for cell detection and nucleus segmentation are novel to the field, whilst the cell boundary segmentation algorithm is contrast-invariant, which makes it more robust on these low-contrast images. Together, this suite of algorithms permit brightfield microscopy image processing without the need for additional fluorescence images. Finally our sephaCe application, which is available at http://www.sephace.com, provides a novel method for integrating these methods with any motorised microscope, thus facilitating the adoption of these techniques in biological research labs.

In Vitro and In Vivo Evaluations of a Hydrophilic 64Cu-Bis(Thiosemicarbazonato)–Glucose Conjugate for Hypoxia Imaging

A water-soluble glucose conjugate of the hypoxia tracer 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) was synthesized and radiolabeled (64Cu-ATSE/A-G). Here we report our initial biological experiments with 64Cu-ATSE/A-G and compare the results with those obtained for 64Cu-ATSM and 18F-FDG. Methods: The uptake of 64Cu-ATSE/A-G and 64Cu-ATSM into HeLa cells in vitro was investigated at a range of dissolved oxygen concentrations representing normoxia, hypoxia, and anoxia. Small-animal PET with 64Cu-ATSE/A-G was performed in male BDIX rats implanted with P22 syngeneic carcinosarcomas. Images of 64Cu-ATSM and 18F-FDG were obtained in the same model for comparison. Results: 64CuATSE/A-G showed oxygen concentration–dependent uptake in vitro and, under anoxic conditions, showed slightly lower levels of cellular uptake than 64Cu-ATSM; uptake levels under hypoxic conditions were also lower. Whereas the normoxic uptake of 64Cu-ATSM increased linearly over time, 64Cu-ATSE/A-G uptake remained at low levels over the entire time course. In the PET study, 64CuATSE/A-G showed good tumor uptake and a biodistribution pattern substantially different from that of each of the controls. In marked contrast to the findings for 64Cu-ATSM, renal clearance and accumulation in the bladder were observed. 64Cu-ATSE/A-G did not display the characteristic brain and heart uptake of 18F-FDG. Conclusion: The in vitro cell uptake studies demonstrated that 64Cu-ATSE/A-G retained hypoxia selectivity and had improved characteristics when compared with 64Cu-ATSM. The in vivo PET results indicated a difference in the excretion pathways, with a shift from primarily hepatointestinal for 64Cu-ATSM to partially renal with 64Cu-ATSE/A-G. This finding is consistent with the hydrophilic nature of the glucose conjugate. A comparison with 18F-FDG PET results revealed that 64Cu-ATSE/A-G was not a surrogate for glucose metabolism. We have demonstrated that our method for the modification of Cu-bis(thiosemicarbazonato) complexes allows their biodistribution to be modified without negating their hypoxia selectivity or tumor uptake properties.

Fluorescent copper(II) bis(thiosemicarbazonates): synthesis, structures, electron paramagnetic resonance, radiolabeling, in vitro cytotoxicity and confocal fluorescence microscopy studies.

Copper bis(4-ethyl-3-thiosemicarbazonato) acenaphthenequinone (1) and copper bis(4-methyl-3-thiosemicarbazonato) acenaphthenequinone (2) are synthesized and characterized in solution, in the solid state, and radiolabeled. Serum-protein binding radioassays show good stability in solution and about 25 % binding to protein over 1 h, which is comparable with the hypoxia selective tracer [(64)Cu(ATSM)]. Cyclic voltammetry shows fast and reversible reduction at redox potentials similar to the values known for hypoxia-selective copper compounds. However, despite this, complex 1 does not show any hypoxic-selective uptake in HeLa cells over 1-h standard assays. Possible reasons for this are studied by using the intrinsic fluorescence of the Cu(II) complexes to determine the cellular distributions and uptake mechanism by confocal microscopy. The complexes are found to bind to the external cell membrane and disperse evenly in the cytoplasm only after a very slow cell internalization (>1 h). No significant changes in distribution are observed by fluorescence imaging under hypoxic conditions. The rate of localization in the cytoplasm contrasts with their Zn(II) analogues, which are known to have fast cell uptake (up to 20 min) and a clear localization in lysosomes and mitochondria. The cytotoxicity mechanism of 1 over 24 h against a number of adherent cell lines is seen to be by membrane disruption and is of a comparable magnitude to that of [Cu(ATSM)], as demonstrated by methyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays.

Bis(thiosemicarbazones) as bifunctional chelators for the room temperature 64-copper labeling of peptides

A range of new carboxylate functionalised bis(thiosemicarbazone) ligands and their Cu(II) complexes have been prepared, fully characterised and radiolabeled in high yield with both (64)Cu and (99m)Tc. Conjugation to a bombesin derivative was achieved using standard solid phase synthetic methodologies and the (64)Cu-labeled conjugate was shown to have good tumour uptake in mice with xenografted PC-3 tumours.

Advanced phase-based segmentation of multiple cells from brightfield microscopy images

Segmenting transparent phase objects, such as biological cells from brightfield microscope images, is a difficult problem due to the lack of observable intensity contrast and noise. Previous image analysis solutions have used excessive de- focusing or physical models to obtain the underlying phase properties. Here, an improved cell boundary detection algorithm is proposed to accurately segment multiple cells within the level set framework. This uses a novel speed term based on local phase and local orientation derived from the monogenic signal, which renders the algorithm invariant to intensity, making it ideal for these images. The new method can robustly handle noise and local minima, and distinguish touching cells. Validation is shown against manual expert segmentations.

New bimetallic compounds based on the bis(thiosemicarbazonato) motif.

Zinc and copper bis(thiosemicarbazonato) complexes containing more than one metal centre have been prepared with a view to examining their application for molecular imaging. The zinc complexes are fluorescent with excitation and emission at relatively long wavelengths. The dinuclear copper complex undergoes two sequential, quasi-reversible reductions.

PHASE-BASED SEGMENTATION OF CELLS FROM BRIGHTFIELD MICROSCOPY

Segmentation of transparent cells in brightfield microscopy images could facilitate the quantitative analysis of corresponding fluorescence images. However, this presents a challenge due to irregular morphology and weak intensity variation, particularly in ultra-thin regions. A boundary detection technique is applied to a series of variable focus images whereby a level set contour is initialised on a defocused image with improved intensity contrast, and subsequently evolved towards the correct boundary using images of improving focus. Local phase coherence is used to identify features within the images, driving contour evolution particularly in near-focus images which lack intensity contrast. Preliminary results demonstrate the effectiveness of this approach in segmenting the main cell body regions

Orthogonal 18F and 64Cu labelling of functionalised bis(thiosemicarbazonato) complexes

The synthesis of three pairs of orthogonally labelled fluorinated Cu bis(thiosemicarbazonato) complexes is presented. These are the first examples of (18)F-labelled Cu(II)-complexes designed to serve as new hypoxia selective PET tracers and as mechanistic probes to study the mode of action of this class of markers. In vitro evaluation revealed that the fluorinated Cu-complex derived from amide coupling is suitable for in vivo work.

Surface functional polymer library by post-polymerisation modification using diarylmethylenes: metal ligand catch and release

Functionalised diarylcarbenes can be used as anchoring agents for the introduction of metal chelating ligands onto the surface of a polystyrene substrate, rapidly creating a library of chelating polymers from a common precursor; although exemplified for polystyrene, the method is potentially applicable to a wide range of polymer substrates. Evaluation of the binding of zinc complexes followed by ligand transfer and release of copper complexes indicated that a pyridyl modified system offered considerable potential for application in the rapid generation of ligand systems of relevance for radiopharmaceutical synthesis.