Section 1 -

Development of In Situ Techniques and Recent Applications Heinz Höfler Instutes of Pathology TUM and Axel Walch GSF, München

Introduction In situ hybridization (ISH) is a technique that allows direct analysis of both genes and gene expression, through the localization of specific nucleic acid sequences to individual cells within a morphologically preserved tissue. The hybridization of nucleic acid probes to their corresponding targets can be used to detect structural changes within the genome (DNA-ISH), or changes in the expression levels of specific transcripts (RNA-ISH) and complement immunohistochemistry studies of protein expression. With the increasing availability of cDNA and cRNA probes to known human gene sequences, hybridization studies can be performed to learn more about regulatory processes and gene expression to diagnose specific diseases and to define the etiology of disease processes. Because this detection can be performed on tissue sections, ISH provides additional morphologic information on the spatial distribution and heterogeneity of gene expression in complex tissues. This offers ISH an advantage over other molecular techniques in which homogenization precludes such evaluation. Additionally this technology can be applied to most routine clinical samples, allowing for the retrospective analysis of abnormalities in genes and gene expression in large numbers of routinely prepared patient biopsy specimens. Although the basic principles of ISH have remained unchanged, high-sensitivity detection, simultaneous assay of multiple species, and automated data collection and analysis have advanced the field significantly. These powerful tools are playing an increasingly important role in both clinical medicine and in translational research. Development of ISH Techniques Early in situ hybridizations depended on radioactive detection, followed in 1977 by the introduction of fluorescently labeled antibodies that recognized specific DNA–RNA hybrids [1]. A more straightforward approach used the chemical coupling of a fluorochrome to a DNA or RNA probe for rapid and direct visualization, known as fluorescence in situ hybridization [2]. The coupling of a fluorochrome to a DNA or RNA probe is often referred to as 'direct labeling'. By contrast, 'indirect labeling' refers to the enzymatic or immunological detection of tags that have been incorporated into a probe. The syntheses of modified nucleotide derivatives that contain a Biotin label, which could be incorporated by polymerases into probes, were instrumental in the development of indirect labeling techniques. During the last few years, several strategies have been developed to improve the sensitivity of ISH by amplification of either target nucleic acid sequences prior to ISH or signal detection after the hybridization is completed [3, 4, 5, 6] . Non-isotopic detection methods are now used routinely for both, DNA-ISH and RNA-ISH. Digoxigenin-, fluorescein-, alkaline phosphatase-, and biotin-labeled probes are most commonly used. Application of some of these techniques has extended the utility of ISH in diagnostic pathology and in research because of the ability to detect targets with low copy numbers of DNA and RNA [5, 6]. Non-isotopically-labeled probes can also be readily applied to ultrastructural RNA-ISH hybridization studies and provide excellent resolution with this technique. Advancing Technology Whereas the initial development of ISH involved expansion of the types of probe and number of detectable targets, the outlook for future development of fluorescence techniques will rather focus the expansion of the subjects of investigation as well as standardization procedures. Clinical application of fluorescence imaging will require further advances in mechanization that allow the probes to be delivered, imaged, and analyzed automatically, thereby reducing operator-to-operator variability. The recently developed tissue microarray (TMA) technology [7] is an ideal platform for the introduction of high-throughput molecular profiling by ISH for tumor specimens at the single cell level. To construct a TMA, small core biopsies are taken from representative areas of paraffin-embedded tumor tissues and assembled in a single block. The numerous advantages of this technology are obvious and have thus stimulated many constructors to evolve and improve different technical approaches. With TMAs, multiple specimens can be simultaneously investigated with different ISH techniques under identical laboratory conditions, resulting in a dramatic time and cost reduction compared with conventional pathologic studies. Furthermore, this technology is less exhausting for the finite original donor material, allowing for a significantly increased number of assays per each case. In addition to pathological specimens such as tumor tissues, microarrays may also contain corresponding normal tissue and internal controls. As the entire group of samples is analyzed simultaneously in one experiment, enormous amounts of correlative information about specific biomarkers at the DNA, RNA or protein level is provided. The next challenge will be to apply multi-parameter ISH technology to these samples to correlate putative genes and their products of prognostic value with specific morphological features initially, and then extend studies to samples where the morphology is not sufficiently informative. Another important development is the increase in the number of differentially labeled probes that can be hybridized and imaged. The discrimination of many more targets than the number of available, spectrally resolvable fluorochromes can be achieved using combinatorial labeling or ratio labeling [8]. The organic fluorophores typically utilized for ISH have relatively broad emission spectra and show significant photobleaching, attributes that limit their use in signal quantification. The recent development of quantum dots (QDs) modified for use in biology provides an important new tool for signal detection in ISH [9]. QD fluorophores have several potential advantages. Their photostability and narrow emission spectra make them eligible candidates for ISH to study the expression of specific mRNA transcripts, furthermore facilitating the analyzing of multiple probes simultaneously. QDs have been successfully used for immunolabeling of cellular molecules, such as HER2 [10] and other cellular targets. Not constricted by problems related to biocompatibility, QDs are now used for in vivo cancer targeting and imaging, particularly when multi-photon imaging is applied. QDs are also well suited for studies in correlative fluorescence and electron microscopy [11]. The use of multi-photon microscopy approaches will also expand application of ISH imaging. In multi-photon microscopy, a laser source fires short bursts of photons that are focused by the microscope to arrive in pairs or triplets such that they summate to excite the fluorophore of interest. Near-infrared excitation light is used, which penetrates biological specimens more deeply and is far less toxic to live samples than visible light. This scheme has already allowed the application of fluorescence imaging to many living systems, including whole animals. Native fluorescent signatures that are present in tissues due to normal physiology or pathophysiological processes can encode important clinical information. Knowledge of how molecules interact in space and time is fundamental for understanding cellular processes. A host of novel techniques have been developed for the visualization of single target molecules in living cells, many based on ISH or immunocytochemistry. To extend the applicability of ISH to living cells, special backbone-modified probes and specific conformations (molecular beacons) have been designed. In the case of IC, conventional immunoreagents have been fine-tuned with respect to size and affinity or replaced with new protein scaffolds based on ankyrin repeat proteins. Other key advances include the use of proximity ligation to confirm vicinity binding and the use of quantum dots, which have proven potential for cellular labeling. Automation, Quantification and Analysis A technical advance in ISH came with the development of machine automation, which could perform on line all of the required incubations in a very precise and controlled fashion, freeing up technician time from what had previously been a very labor intensive procedure. Automation allows for rapid and easy optimization of variables such as probe concentration, hybridization temperature, reaction times, and enzymatic permeabilization of tissue - all critical variables in successful ISH. Automation coupled with improvements in detection of hybridization and the ready availability of nucleic acid probes has made ISH more sensitive, consistent, reliable and feasible than ever before, with high-throughput capabilities. There are several commercially available automated in situ hybridization machines that would greatly improve the throughput as well as the reproducibility. These advances have highly expanded the application of ISH in the study of both, the genome (DNA-ISH), or changes in the expression levels of specific transcripts (RNA-ISH) in routine clinical samples as well as in basic science research. By automation, the drawbacks of manual quantification and analysis of ISH can be circumvented. Because fluorescent signal counting is tedious and often subjective, automated digital algorithms for calculating signals are desirable. Objective criteria may be introduced, which are followed without bias of the investigator or reduction of efficiency due to fatigue. The number of cells detected may be increased without significantly increasing manual workload. Up to date, most publications regarding automated ISH analysis report assessment of amplification of genes or enumeration of centromeric probes. Some reports describe analyses of translocations applying locus specific probes. An automated analysis follows the steps of manual FISH analysis. Cell nuclei must be correctly recognized. Nuclear debris, large clusters of overlapping or touching nuclei, or unspecific background staining must be excluded from further analysis, even if this exclusion results in the loss of a few correctly acquired cells. Signals must be correctly detected. Since intra- and internuclear variation regarding shape, size, and intensity of FISH signals is significant, it is not possible to differentiate signals from artifacts, background noise, split, or partially overlapping signals in every case. Therefore, it is crucial to exclude incorrectly recognized signals and cells with aberrant signal patterns. Since nuclei are three-dimensional (3D) objects, measuring in 3D is vital to reduce the false positive rate resulting from the overlap of signals in the 2D projection of the nuclei. New technology provides a stack of images on multiple focal planes throughout a tissue sample. Multiple-focal-plane imaging helps overcome the biases and imprecision inherent in single-focal-plane methods. ISH for Studying the Genome in Three Dimensions In three-dimensional fluorescence in situ hybridization (3D-FISH) nuclei are fixed so that the spatial relationships between chromosome territories are maintained. Sophisticated 3D image-acquisition technology is used to collect a series of images throughout different sections of the nucleus, which allows a detailed three-dimensional reconstruction. 3D-FISH has greatly benefited from improvements in imaging technologies that use confocal lasers or sophisticated deconvolution algorithms. Interphase FISH, both on fixed nuclei and in living cells, allows the study of the functional organization of the genome and the dynamic interplay between the genome and its regulatory factors. Studies of chromosomes within their natural tissue context using 3D-FISH have allowed the study of higher-order chromatin architecture [12]. In most tissues, the radial arrangement of chromosome territories has been shown to correlate with gene density. However, in fibroblasts the distribution of chromosome territories correlates more closely with chromosome size, indicating that there are tissue-specific differences in three-dimensional genome organization. Three-dimensional studies are also providing increasing evidence that chromatin location within the nucleus is an important constraint on gene activity. Recent 3D-FISH studies indicated that portions of different chromosomes interact with each other, which implies that related genes are brought together in the nucleus to coordinate their expression [12]. However, these technologies currently lack the resolution and speed that is required for the clinical diagnosis of structural rearrangements and the instrumentation that is required is expensive. Nevertheless, analyses of higher-order chromatin structure by 3D-FISH will be of importance in the future. New studies should address distinct three-dimensional organizations in different tissue types, as higher-order chromatin arrangements are likely to have fundamental implications for development and cell differentiation. Conclusion In situ hybridization, the method of choice for localization of specific nucleic acid sequences in the native cellular or histological context, is a 20-year-old technology that has developed continuously. Various methodologies and modifications have been introduced to optimize the detection of DNA and RNA. Although the basic principles of ISH have remained unchanged, high-sensitivity detection, simultaneous assay of multiple species, and automated data collection and analysis have advanced the field significantly. In the future, ISH techniques are likely to have significant further impact on live-cell imaging and on medical diagnostics. References Rudkin, G. T. & Stollar, B. D. High resolution detection of DNA−RNA hybrids in situ by indirect immunofluorescence. Nature 1977;265, 472−473. Bauman, J. G., Wiegant, J., Borst, P. & van Duijn, P. A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochromelabelled RNA. Exp Cell Res.1980;128, 485−490. Mueller JD , Putz B, Hofler H.: Self-sustained sequence replication (3SR): an alternative to PCR. Histochem Cell Biol. 1997;108(4-5):431-7. Hoefler H , Childers H, Montminy MR, Lechan RM, Goodman RH, Wolfe HJ.: In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes. Histochem J. 1986;18(11-12):597-604. Hoefler H.: What's new in "in situ hybridization". Pathol Res Pract. 1987;182(3):421-30. Qian X , Lloyd RV.: Recent developments in signal amplification methods for in situ hybridization. Diagn Mol Pathol. 2003;12(1):1-13. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP.:Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998;4(7):844-7. Speicher MR, Carter NP. The new cytogenetics: blurring the boundaries with molecular biology. Nat Rev Genet. 2005;6(10):782-92. Chan WC, Maxwell DJ, Gao X, Bailey RE, Han M, Nie S. Luminescent quantum dots for multiplexed biological detection and imaging. Curr Opin Biotechnol. 2002;13(1):40-6. Wu X, Liu H, Liu J, Haley KN, Treadway JA, Larson JP, Ge N, Peale F, Bruchez MP. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol. 2003;21(1):41-6. Giepmans BN, Adams SR, Ellisman MH, Tsien RY. The fluorescent toolbox for assessing protein location and function. Science. 2006;312(5771):217-24. Cremer, T. & Cremer, C. Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nature Rev Genet 2001; 2, 292−301.