Antti Seppo

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

Detection of circulating tumor cells in peripheral blood with an automated scanning fluorescence microscope

We have developed an automated, highly sensitive and specific method for identifying and enumerating circulating tumour cells (CTCs) in the blood. Blood samples from 10 prostate, 25 colorectal and 4 ovarian cancer patients were analysed. Eleven healthy donors and seven men with elevated serum prostate-specific antigen (PSA) levels but no evidence of malignancy served as controls. Spiking experiments with cancer cell lines were performed to estimate recovery yield. Isolation was performed either by density gradient centrifugation or by filtration, and the CTCs were labelled with monoclonal antibodies against cytokeratins 7/8 and either AUA1 (against EpCam) or anti-PSA. The slides were analysed with the Ikoniscope® robotic fluorescence microscope imaging system. Spiking experiments showed that less than one epithelial cell per millilitre of blood could be detected, and that fluorescence in situ hybridisation (FISH) could identify chromosomal abnormalities in these cells. No positive cells were detected in the 11 healthy control samples. Circulating tumour cells were detected in 23 out of 25 colorectal, 10 out of 10 prostate and 4 out of 4 ovarian cancer patients. Five samples (three colorectal and two ovarian) were analysed by FISH for chromosomes 7 and 8 combined and all had significantly more than four dots per cell. We have demonstrated an Ikoniscope® based relatively simple and rapid procedure for the clear-cut identification of CTCs. The method has considerable promise for screening, early detection of recurrence and evaluation of treatment response for a wide variety of carcinomas.

Inactivation of the Mgat1 gene in oocytes impairs oogenesis, but embryos lacking complex and hybrid N-glycans develop and implant.

ABSTRACT Complex and hybrid N-glycans contain sugar residues that have been implicated in fertilization, compaction of the embryo, and implantation. Inactivation of the Mgat1 gene responsible for their synthesis is embryonic lethal, but homozygous mutant blastocysts are phenotypically normal due to the presence of maternal Mgat1 gene transcripts. To identify roles for complex and hybrid N-glycans in oogenesis and preimplantation development, the Mgat1 gene in oocytes was deleted by using a ZP3Cre recombinase transgene. All mutant oocytes had an altered zona pellucida (ZP) that was thinner than the control ZP, and they did not possess complex N-glycans but contained ZP1, ZP2, and ZP3 glycoproteins. Mutant eggs were fertilized, all embryos implanted, and heterozygotes developed to birth. However, mutant females had decreased fertility, yielded fewer eggs after stimulation with gonadotropins, and produced a reduced number of preimplantation embryos and less progeny than controls. About 25% of embryonic day 3.5 (E3.5) embryos derived from mutant eggs were severely retarded in development, even when they were heterozygous and expressed complex N-glycans. Thus, a proportion of Mgat1− / − oocytes were developmentally compromised. Surprisingly, mutant eggs also gave rise to Mgat1 − / − embryos that developed normally, implanted, and progressed to E9.5. Therefore, complex or hybrid N-glycans are required at some stage of oogenesis for the generation of a developmentally competent oocyte, but fertilization, blastogenesis, and implantation may proceed in their absence.

Induction of neuron-specific glycosylation by Tollo/Toll-8, a Drosophila Toll-like receptor expressed in non-neural cells.

Specific glycan expression is an essential characteristic of developing tissues. Our molecular characterization of a mutation that abolishes neural-specific glycosylation in the Drosophila embryo demonstrates that cellular interactions influence glycan expression. The HRP epitope is an N-linked oligosaccharide expressed on a subset of neuronal glycoproteins. Embryos homozygous for the TM3 balancer chromosome lack neural HRP-epitope expression. Genetic and molecular mapping of the relevant locus reveals that Tollo/Toll-8, a member of the Toll-like receptor family, is altered on the TM3 chromosome. In wild-type embryos, Tollo/Toll-8 is expressed by ectodermal cells that surround differentiating neurons and precedes HRP-epitope appearance. Re-introduction of Tollo/Toll-8 into null embryos rescues neural-specific glycan expression. Thus, loss of an ectodermal cell surface protein alters glycosylation in juxtaposed differentiating neurons. The portfolio of expressed oligosaccharides in a cell reflects its identity and also influences its interactions with other cells and with pathogens. Therefore, the ability to induce specific glycan expression complements the previously identified developmental and innate immune functions of Toll-like receptors.

Gain of 3q26: A genetic marker in low-grade squamous intraepithelial lesions (LSIL) of the uterine cervix

OBJECTIVE Physicians have few resources for determining which LSIL will progress to HSIL or regress. Recently the chromosome 3q26 region was found to be amplified in patients with cervical cancer. The frequency of this 3q gain increased with severity of dysplasia. The primary objective of this study was to evaluate an automated FISH assay for detection of 3q gain in liquid cytology samples as a potential tool for risk stratification and triaging. METHODS Slides prepared from 257 liquid cytology specimens (97 Negative, 135 LSIL 25 HSIL) were hybridized with a single-copy probe for the chromosome 3q26 region and a probe for the centromeric alpha-repeat sequence of chromosome 7, using standard FISH methods. Using automated analysis, the total number of nuclei and the number of nuclei with >2 signals for 3q26 were determined, using a 20x objective. The nuclei were rank ordered based on number of 3q26 FISH signals. The 800 nuclei with the highest number of signals were scored using both FISH probes and nuclei with increased numbers of 3q signals were enumerated. RESULTS AND CONCLUSIONS Analysis of 257 specimens demonstrated that a fully automated FISH scoring system can detect 3q gain in liquid cytology samples. A fully automated method for determination of 3q gain in liquid cytology may be the assay necessary to implement routine testing. Additional studies to validate the utility of this technology are needed.

Detection of circulating fetal cells utilizing automated microscopy: potential for noninvasive prenatal diagnosis of chromosomal aneuploidies

As fetal cells can be indisputably identified through detection of Y FISH signals, we utilized an automated microscopy system developed to identify and enumerate cells bearing X and Y FISH signals. We further investigated the potential of fetal hemoglobin expression as a gender independent marker for automated identification of fetal cells.

POWER WITHOUT INFLUENCE? THE EU AND TRADE DISPUTES WITH RUSSIA

THE EU HAS BEEN VARIOUSLY DESCRIBED AS A GLOBAL POWER, a superpower, a civilian power, a trade power, a normative power, a realist power and an ethical power, but it remains unclear when and how the EU really can exercise its power effectively. The gap between expectations and capabilities in the EU external relations is well known (Hill 1993). Perhaps it has nowhere manifested itself as clearly as in the EU’s relations with Russia. A recent ‘Power Audit of EU–Russia Relations’ conducted by the European Council of Foreign Relations (ECFR) finds that the EU has hardly been able to influence Russia although it is a far bigger power than Russia in conventional terms (Leonard & Popescu 2007). According to the ECFR report the main explanation for the inability of the EU to influence Russia is its lack of unity. The problem of the insufficient cohesion of the EU is not a novel finding (Allen 1997; Meunier & Nicolaidis 1999; Smith 2001a; Toje 2008). Yet, it has become more common to refer to it for two reasons especially in the context of contemporary EU–Russian relations: firstly, the EU has enlarged to become a community of 27 member states; secondly, Russia has started effectively to use a divide-and-rule strategy vis-à-vis the EU (Haukkala 2006). Yet the lack of unity does not need to be the only explanation for the EU’s poor influence. The ECFR also points out that perhaps, even where it has been united, the EU has not been able to choose the best possible strategy or to implement it properly to achieve its aims. The EU does not often rely on hard military and economic power even if it has such hard power resources available, but tends to prefer inducements, persuasion, invoking norms and acting as on example: power tools that are often associated with the EU’s identity as a normative power (Manners 2002). It may also use its policy instruments in an incoherent way and so fail to achieve optimal effectiveness. Finally, the whole idea that the EU should prevail in trade disputes with Russia just because the EU has more economic leverage might be misleading. It might be unrealistic to expect the EU to be able change the position of Russia on issues where Russia has important defensive stakes, even if the EU were united and had adopted the best possible strategy. This article will focus on the capabilities–expectations gap in the EU’s Russia policy. In order to avoid simplistic conclusions, a distinction will be made between the EUROPE-ASIA STUDIES Vol. 61, No. 10, December 2009, 1805–1823

Enzyme-assisted synthesis of a bivalent high-affinity dodecasaccharide inhibitor of mouse gamete adhesion The length of the chains carrying distal α1,3-bonded galactose residues is critical

Proposing to study the molecular mechanisms of mouse gamete adhesion with the aid of high affinity adhesion inhibitors of saccharide nature, we report here the enzymatic synthesis of a bivalent oligosaccharide Galα1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3(Galα1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐6)Galβ1‐4GlcNAc (4), consisting of two long arms that link together two distal α1,3‐galactose residues. Binding data reported elsewhere (E. Litscher et al., Biochemistry, 1995, 34, 4662–4669) show that 4 is a high affinity inhibitor of mouse gamete adhesion in vitro (IC50 = 9 μM), while a related octasaccharide Galα1‐3Galβ1‐4GlcNAcβ1‐3(Galα1‐3Galβ1‐4GlcNAcβ1‐6)Galβ1‐4GlcNAc, consisting of two short arms is of very low inhibitory activity. The data highlight the importance of the two α‐galactose residues of 4, and the length of the sugar chains joining them.

Oligo-N-acetyllactosaminoglycans bearing Galβ1-4(Fucα1-3)GlcNAc sequences reveal lower affinities than their nonfucosylated, or α(1-2) fucosylated counterparts for immobilized wheat germ agglutinin

Relative affinities of several fucosylated and nonfucosylated oligo-N-acetyllactosaminoglycans for immobilized wheat germ agglutinin (WGA) were studied using a chromatographic technique. α(1-3) Fucosylation of theN-acetylglucosamine unit(s) in mono- and biantennary saccharides of the Galβ1-4GlcNAc-R type strongly reduced the WGA-affinity. In contrast, α(1-2) fucosylation of the nonreducing galactose unit(s) of the saccharides did not reduce the affinity.

Construction of linear GlcNAcβ1-6Galβ1-OR type oligosaccharides by partial cleavage of GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-OR sequences with jack bean β-N-acetylhexosaminidase

Radiolabelled GlcNAcβ1-3(GlcNAcβ1-6)Gal (1), GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-OCH3 (4), GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4Glc (7), and GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc (10) were cleaved partially with jack bean β-N-acetylhexosaminidase (EC 3.2.1.30), and the digests were analysed chromatographically. All four oligosaccharides were hydrolysed faster at the (1-6) branch, than at the (1-3) branch, but a high branch specificity was observed only with the glycan4. The saccharides1 and7 resembled each other in the kinetics of the enzyme-catalysed release of their two non-reducingN-acetylglucosamine units, but the glycan10 was rather different. The partial digestions made it possible to obtain radiolabelled GlcNAcβ1-6Gal, GlcNAcβ1-6Galβ1-OCH3, GlcNAcβ1-6Galβ1-4Glc, and, in particular, GlcNAcβ1-6Galβ1-4GlcNAc.