Tissue microarray profiling of cancer specimens and cell lines: opportunities and limitations

The implementations of high-throughput genetic technologies, such as oligonucleotide microarrays, generate myriad points of data. The identified potential candidate genes need to be further characterized and selected using a large number of well-characterized tumors and stringent criteria. Tissue microarrays allow for such high-throughput expression profiling of tumor samples, providing, in addition, information at the microanatomical level. Different techniques could be applied for identification of specific phenotypic (immunohistochemistry and in situ hybridization) or genotypic (fluorescence in situ hybridization) alterations, holding strong potential for translational research. Tissue microarrays consisting of 0.6-mm biopsies of paraffin-embedded tissues are well validated and have been used for various clinicopathological studies. This review discusses the technical considerations for construction of such arrays from paraffin-embedded tissues and cell lines and outlines their potential for clinical research applications. The use of paraffin-embedded tissues has some limitations with regard to analysis of RNA or certain proteins. To overcome such limitations, we have developed a cryoarray strategy allowing for the processing of multiple frozen tissue specimens and/or cell lines on a single tissue block. These approaches offer the opportunity to conduct pilot and validation studies of potential targets using clinical samples linked to clinicopathological databases.     

Quantitative cell imaging using single beam phase retrieval method

Quantitative three-dimensional imaging of cells can provide important information about their morphology as well as their dynamics, which will be useful in studying their behavior under various conditions. There are several microscopic techniques to image unstained, semi-transparent specimens, by converting the phase information into intensity information. But most of the quantitative phase contrast imaging techniques is realized either by using interference of the object wavefront with a known reference beam or using phase shifting interferometry. A two-beam interferometric method is challenging to implement especially with low coherent sources and it also requires a fine adjustment of beams to achieve high contrast fringes. In this letter, the development of a single beam phase retrieval microscopy technique for quantitative phase contrast imaging of cells using multiple intensity samplings of a volume speckle field in the axial direction is described. Single beam illumination with multiple intensity samplings provides fast convergence and a unique solution of the object wavefront. Three-dimensional thickness profiles of different cells such as red blood cells and onion skin cells were reconstructed using this technique with an axial resolution of the order of several nanometers.     

A new method to make nuclei or cell microarrays.

We invented a new method to make microarrays using nuclei extracted from paraffin-embedded tissues or cultured cells. A blank recipient paraffin block with 10 x 10 cores was constructed and sectioned to make the mold for the cell arrays. The sections of paraffin were mounted on poly-L-lysine-coated slides. Prepared nuclei or cells were injected into the cores of the paraffin mold. The slides were dried and dewaxed and nuclei or cell arrays were made. Using this method, we successfully made microarrays of nuclei extracted from diffuse large B-cell lymphoma paraffin-embedded tissues, nasopharyngeal cancer and lymphoma cell lines. This technique resulted in a paraffin-embedded cell preparation that yielded a cell density of approximately 500 to 1000 or 800 cells on average per 0.6-mm-diameter core. The microarrays were successfully used in fluorescence in situ hybridization, mRNA in situ hybridization, and cytohistochemical staining.     

Modulation of expression of intermediate filaments during the development of established rat liver cell lines

A number of clear epithelial-like cell lines were established from the liver of fetal and neonatal rats. The intermediate filaments of these cells were investigated using polyclonal prekeratin antisera, monoclonal antibodies against a cytokeratin subfamily and vimentin by means of the indirect immunofluorescence technique and SDS-PAGE analysis. Changes in the expression of cytokeratin filaments were found during the evolution of permanent cell lines. Cells positively stained for cytokeratins could be seen near to other cells which were negative. In early passages (up to the 40th) nearly all cells were strongly stained by the different keratin antibodies. During the following subcultivation the pattern of staining considerably changed. In FRL and NP-RL cell lines keratin-negative cells could already be observed in the early passages, rapidly increasing in the later passages. Compared to this, vimentin-staining of all cells remained constant in its morphological expression. The keratin filaments were seen in thick fiber bundles arranged particularly in the perinuclear ring as well as in finer networks throughout the cytoplasm. Every cell in the established lines showed their very individual staining pattern. The vimentin filaments extended to the whole cytoplasm up to the cell margin. Our observations demonstrate the variability of the system of keratin filaments in established epithelial-like liver cells under cultural conditions.     

Spectrum of genetic changes in gastro-esophageal cancer cell lines determined by an integrated molecular cytogenetic approach

Adenocarcinomas arising around the gastro-esophageal junction (GEJ) are highly malignant, and their incidence has risen rapidly in the last decades. Cell lines are the basic in vitro system for functional and therapeutic studies in GEJ tumors, but only a small number of cell lines are currently available, and none of them has been fully karyotyped. We analyzed 5 GEJ tumor cell lines using a combination of 24-color fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH) and genomic microarrays. Using CGH we demonstrated that these cell lines present imbalances similar to those we had previously observed in primary GEJ tumors, namely gains on 1q, 7q, 8q, 17q, 19q, 20, and X, and losses on 3p, 4, 5q, 9p, 18q, and 21. Multicolor FISH karyotyping revealed multiple structural rearrangements involving chromosomes 1, 5, 6, 7, 8, 9, 13, 17, 18, and 22. Rearrangements of chromosome 8 involved 10 different chromosomes, while rearrangements of chromosome 17 involved 5. Different rearrangements resulted in imbalances of similar chromosome regions, suggesting that similar genomic imbalances are constitutively important but are achieved through different pathways. The use of a commercially available genomic array excluded TOP2A (17q), and MYBL2, PTPT1, CSE1L, and ZNF217 (20q) as candidate genes for frequently amplified areas on these chromosomes, and contributed to refining the limits of chromosome regions involved in genomic imbalances.     

An image-processing technique for measuring the dynamic movement of cell membranes

An automated method has been developed to measure and compare the dynamic movement of cell membranes. Using the red blood cell as a common example the method locates the edge of the cell, with sub-pixel precision at multiple points on the periphery. This method is a different implementation to a technique used for giant unilamellar vesicles and addresses issues relating specifically to biological cells, in particular relating to finding a local minima, calculating equi-spaced measuring points for arbitrary shapes and using the perpendicular direction to the edge for position measurement. Parameters have been defined to characterise the cell's membrane behaviour and the analysis program allows the automatic compilation of multiple tests under varying conditions, and statistical comparison of identical populations of cells.     

Micropatterned surfaces for controlling cell adhesion and rolling under flow

Cell adhesion and rolling on the vascular wall is critical to both inflammation and thrombosis. In this study we demonstrate the feasibility of using microfluidic patterning for controlling cell adhesion and rolling under physiological flow conditions. By controlling the width of the lines (50–1000 μm) and the spacing between them (50–100 μm) we were able to fabricate surfaces with well-defined patterns of adhesion molecules. We demonstrate the versatility of this technique by patterning surfaces with 3 different adhesion molecules (P-selectin, E-selectin, and von Willebrand Factor) and controlling the adhesion and rolling of three different cell types (neutrophils, Chinese Hamster Ovary cells, and platelets). By varying the concentration of the incubating solution we could control the surface ligand density and hence the cell rolling velocity. Finally by patterning surfaces with both P-selectin and von Willebrand Factor we could control the rolling of both leukocytes and platelets simultaneously. The technique described in this paper provides and effective and inexpensive way to fabricate patterned surfaces for use in cell rolling assays under physiologic flow conditions.     

Rapid characterization of human chromosomes in hybrid cell lines by primed in situ (PRINS) labeling

The primed in situ (PRINS) labeling technique allows a rapid and specific labeling of human chromosomes in situ. This method is based on annealing of specific oligonucleotide primers and subsequent primer extension by a Taq DNA polymerase. We have developed a PRINS protocole for the cytogenetic analysis of somatic hybrid cell lines. Painting of human chromosomes is performed using Alu specific primers. Individual human chromosomes are identified using chromosome-specific alpha-satellite primers. The method was successfully tested to 3 different human-hamster hybrid cell lines. This approach provides an interesting alternative to classical cytogenetic and in situ hybridization techniques for the characterization of the human content of hybrid cell lines.     

Comparison of gene expression between metastatic derivatives and their poorly metastatic parental cells implicates crucial tumor-environment interaction in metastasis of head and neck squamous cell carcinoma

Metastasis of human head and neck cancer is a multistep and highly heterogeneous process requiring activation and deactivation of multiple and specific genes. To identify these genes, we established highly metastatic head and neck squamous cell carcinoma (HNSCC) cell lines from poorly metastatic HNSCC cells through in vivo selection using a lymph node metastatic mouse model. The very close genetic relationship between these highly metastatic cell lines and the parental cell line provided an excellent model for differential gene expression analysis using cDNA microarrays. Comparison of 6 cell lines established individually from the lymph node metastases with their poorly metastatic parental cell line revealed 33 differentially expressed genes. Some of these genes are involved in cellular signal transduction and matrix modeling. Differences in expression of members of the tumor necrosis factor, interleukin, caspase, and matrix metalloproteinase families were also examined. We found that two upregulated genes participated in the NF-κB regulatory pathway. Furthermore, differences in gene expression between six cell lines derived from primary tumors and six cell lines derived from lymph node metastases in the mouse model were analyzed statistically. Tissue growth factor-β and tumor necrosis factor-related genes showed significantly altered expression in cells derived from lymph node metastases as compared with cells derived from primary tumors, suggesting that the differential growth advantage of metastatic cells requires more aggressive responses to their environment, such as a lymph node tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

RNA analysis of B cell lines arrested at defined stages of differentiation allows for an approximation of gene expression patterns during B cell development

The development of a mature B lymphocyte from a bone marrow stem cell is a highly ordered process involving stages with defined features and gene expression patterns. To obtain a deeper understanding of the molecular genetics of this process, we have performed RNA expression analysis of a set of mouse B lineage cell lines representing defined stages of B cell development using Affymetrix microarrays. The cells were grouped based on their previously defined phenotypic features, and a gene expression pattern for each group of cell lines was established. The data indicated that the cell lines representing a defined stage generally presented a high similarity in overall expression profiles. Numerous genes could be identified as expressed with a restricted pattern using dCHIP-based, quantitative comparisons or presence/absence-based, probabilistic state analysis. These experiments provide a model for gene expression during B cell development, and the correctly identified expression patterns of a number of control genes suggest that a series of cell lines can be useful tools in the elucidation of the molecular genetics of a complex differentiation process.     

Expression of intermediate filaments in established human lung cancer cell lines. An indicator of differentiation and derivation

A panel of established human pulmonary cancer cell lines, representing the major histopathologic groups according to the World Health Organization (WHO) classification (WHO 1, squamous cell carcinoma; WHO 2, small cell carcinoma; WHO 3, adenocarcinoma; WHO 4, large cell carcinoma) were examined for their expression of various types of intermediate filaments in order to determine their phenotypic differences and to attempt to disclose their histogenetic origin. The cells were investigated with antibodies specific for cytokeratin, vimentin, and neurofilament polypeptides with both immunofluorescence microscopy and immunoblotting techniques. Squamous cell carcinoma and adenocarcinomas expressed cytokeratin in accordance with the epithelial nature of these tumors but not neurofilament polypeptides. Small cell carcinomas, on the other hand, were positive for neurofilaments but negative for keratin. In contrast to small cell carcinoma, adenocarcinoma, and squamous cell carcinoma, one cell line derived from large cell carcinoma appeared to express both neurofilaments and keratin. All cell lines studied also contained variable amounts of vimentin, a phenotypic characteristic obtained by many cells under in vitro conditions. The results demonstrate, in accordance with our earlier observations in vivo, a distinctly divergent expression of intermediate filament proteins in different types of lung cancers. The persistence of this phenotypic heterogeneity in vitro consolidates the use of cell cultures as useful models to study the biologic behavior and interrelationships of lung cancers. Based on the present studies, and taking into account the occurrence of mixed forms of lung cancers, we present a hypothetical scheme of the histogenetic derivation of different types of lung cancers.     

A matrix micropatterning platform for cell localization and stem cell fate determination

To study the role of cell–extracellular matrix (ECM) interactions, microscale approaches provide the potential to perform high throughput assessment of the effect of the ECM microenvironment on cellular function and phenotype. Using a microscale direct writing (MDW) technique, we characterized the generation of multicomponent ECM microarrays for cellular micropatterning, localization and stem cell fate determination. ECMs and other biomolecules of various geometries and sizes were printed onto epoxide-modified glass substrates to evaluate cell attachment by human endothelial cells. The endothelial cells displayed strong preferential attachment to the ECM patterned regions and aligned their cytoskeleton along the direction of the micropatterns. We next generated ECM microarrays that contained one or more ECM components (namely gelatin, collagen IV and fibronectin) and then cultured murine embryonic stem cell (ESCs) on the microarrays. The ESCs selectively attached to the micropatterned features and expressed markers associated with a pluripotent phenotype, such as E-cadherin and alkaline phosphatase, when maintained in growth medium containing leukemia inhibitory factor. In the presence of the soluble factors retinoic acid and bone morphogenetic protein-4 the ESCs differentiated towards the ectodermal lineage on the ECM microarray with differential ECM effects. The ESCs cultured on gelatin showed significantly higher levels of pan cytokeratin expression, when compared with cells cultured on collagen IV or fibronectin, suggesting that gelatin preferentially promotes ectodermal differentiation. In summary, our results demonstrate that MDW is a versatile approach to print ECMs of diverse geometries and compositions onto surfaces, and it is amenable to the generation of multicomponent ECM microarrays for stem cell fate determination.     

A comparison of methods for the detection of human papillomavirus DNA by in situ hybridization with biotinylated probes on human carcinoma cell lines. Application to wart sections

We compared nine different techniques for the detection of biotinylated DNA-DNA HPV hybrids on HeLa cells with 10-50 copies of HPV 18 DNA per cell. CaSki cells with 600 copies of HPV 16 DNA per cell and tissue sections from frozen or paraffin-embedded biopsy specimens. The cell samples were either cell deposits or cytocentrifuged or cultured slides. In most cases, the samples (cell deposits and tissue sections) were denatured with hybridization mixture prepared under stringent conditions (Tm = -17 degrees C) containing biotinylated DNA probes (cloned HPV types 1, 2, 6, 11, 16 and 18), at 90 degrees C for 10 min. In other cases (cytocentrifuged or cultured cells), the denaturation was performed by HCl hydrolysis and mild heating at 50 degrees C; the probes were denatured separately by heating. All the samples were further incubated overnight at 37 degrees C. For HPV DNA detection, three amplification levels were used on cell deposits. Only the techniques involving a three-step reaction (a rabbit anti-biotin antibody - a biotinylated goat anti-rabbit antibody - a complex of streptavidin-alkaline phosphatase or streptavidin-gold or streptavidin-fluorescein) gave satisfactory results, on both cell lines. With the one step reaction (an avidin-horseradish peroxidase, or streptavidin-alkaline phosphatase or streptavidin-fluorescein complex), no labeling of HeLa cells was observed with any of the HPV probes, including HPV 18. The techniques involving four steps (avidin or streptavidin - anti-avidin goat antibody or anti-streptavidin rabbit antibody - a biotinylated anti-goat (or anti-rabbit) antibody - a complex of avidin-biotin-peroxidase or streptavidin-biotin-alkaline phosphatase or streptavidin-biotin-horseradish peroxidase) resulted in high background on both cell lines. For the reproducible detection of low copy number of HPV DNA (less than 50 copies) such as occur in HeLa cells our data suggested that the three-step technique with the streptavidin-alkaline phosphatase complex was the method of choice. The most intense labeling was always obtained with cell deposits and the technique was successfully applied to frozen and paraffin-embedded tissue sections from typical warts.     

MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells

Many of the currently established human embryonic stem (hES) cell lines have been characterized extensively in terms of their gene expression profiles and genetic stability in culture. Recent studies have indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Using both microarrays and quantitative PCR, we report here the differences in miRNA expression between undifferentiated hES cells and their corresponding differentiated cells that underwent differentiation in vitro over a period of 2 weeks. Our results confirm the identity of a signature miRNA profile in pluripotent cells, comprising a small subset of differentially expressed miRNAs in hES cells. Examining both mRNA and miRNA profiles under multiple conditions using cross-correlation, we find clusters of miRNAs grouped with specific, biologically interpretable mRNAs. We identify patterns of expression in the progression from hES cells to differentiated cells that suggest a role for selected miRNAs in maintenance of the undifferentiated, pluripotent state. Profiling of the hES cell "miRNA-ome" provides an insight into molecules that control cellular differentiation and maintenance of the pluripotent state, findings that have broad implications in development, homeostasis, and human disease states.     

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines

Summary.  We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef-6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV. Received May 3, 2002; accepted September 19, 2002     

Programmed cell death (apoptosis) in lymphoid and myeloid cell lines during zinc deficiency.

Three human cell lines of lymphoid (Molt-3 and Raji) or myeloid (HL-60) origin were maintained in vitro under zinc-sufficient or zinc-deficient conditions. Under these conditions, cell proliferation, viability and mode of death (apoptotic or necrotic) were assessed. All three cell types decreased their proliferative capacity and viability under conditions of zinc deficiency. Cell death in the HL-60 and Raji cultures occurred primarily via apoptosis, while most cells in zinc-deficient Molt-3 cultures died via necrosis. Apoptosis in zinc-deficient cultures of HL-60 and Raji cells was characterized by a slow decline in culture viability as cells with condensed and fragmented nuclear DNA appeared. These morphological changes were accompanied by an increase in cell buoyant density, which allowed separation of viable apoptotic cells from their non-apoptotic counterparts by means of percoll stepdensity gradients. Necrosis in zinc-deficient Molt-3 cultures was characterized by rapid loss of cell culture viability as these cells underwent direct lysis. Intact necrotic cells were easily identified by the flocculated state of their chromatin as well as the decreased basophilia of their cytoplasm. Analysis of DNA from apoptotic HL-60 and Raji cells revealed that internucleosomal DNA degradation, indicative of endogenous endonuclease activation, had occurred, whereas the nuclear DNA of necrotic Molt-3 cells remained relatively unfragmented. The different modes of cell death evoked may reflect the relative sensitivities of cells of these lineages to zinc levels in vivo.     

Study of cell deformability by a simple method

Cell deformability plays an important role in many immunological processes, such as phagocyte chemotaxis and endocytosis. The most widely used method of assay consists in aspirating cells into glass micropipettes and measuring the length of the protrusion induced by a given pressure, or the minimum pressure required to drive cells into the micropipette. This procedure requires specialized equipment and delicate manipulation. The present report describes a simpler procedure: cells are centrifuged in petri dishes floating on a water cushion, then fixed and coated with 0.8 μm diameter latex beads, which allows rapid and accurate determination of their height. This method is compared with the micropipette technique by studying lymphocyte and macrophage-like cell lines in physiological medium and in the presence of a divalent cation chelator or a microfilament inhibitor. In addition to simplicity, the main advantages of this technique are that (i) many cells may be examined within a reasonable period of time, which allows testing of heterogenous cell populations, and (ii) unexpectedly, centrifugation was quite harmless under our experimental conditions, since it did not impair cell proliferative ability nor phagocytic ability.It is concluded that the method may be used in clinical laboratories to explore phagocyte dysfunctions, as well as in experimental studies.     

Tissue Microarrays: Applications in Neuropathology Research, Diagnosis, and Education

Tissue microarrays (TMAs) are composite paraffin blocks constructed by extracting cylindrical tissue core "biopsies" from different paraffin donor blocks and re-embedding these into a single recipient (microarray) block at defined array coordinates. Using this technique, up to 1000 or more tissue samples can be composited into a single paraffin block. Tissue microarrays permit high-volume simultaneous analysis of molecular targets at the DNA, mRNA, and protein levels under identical, standardized conditions on a single glass slide, and also provide maximal preservation and utilization of limited and irreplaceable archival tissue samples. This versatile technique facilitates retrospective and prospective human tissue studies, animal tissue studies, and cell line cytospin cell block studies. In this review, we present the technical aspects of TMA construction and sectioning, validation aspects of the technique, TMA advantages and limitations, and a sampling of the broad range of TMA uses in modern neuropathologic clinical diagnosis, research, and education. A specific illustration of the most widely employed and increasingly important TMA application is also presented: confirmation via TMA-based immunohistochemistry of the differential expression of a marker (IGFBP2) initially identified by gene expression profiling to be overexpressed in glioblastoma.     

Validation of tissue microarrays using p53 immunohistochemical studies of squamous cell carcinoma of the larynx.

Tissue microarrays are a powerful new tissue-conserving technology in the study of cancer, allowing simultaneous study of a large number of tumor specimens. We sought to ascertain the utility of tissue microarrays in head and neck cancer pathology using squamous cell carcinoma of the larynx as a model system. Whole-specimen slides from 44 different laryngeal squamous cell carcinomas were stained for p53 expression. Microarrays were then generated by taking six 0.6-mm core biopsies from each of the 44 specimens. The whole sections and the microarrays were independently scored for p53 expression. Twenty-three (53%) of the 44 tumor specimens were positive for p53. Forty-four of the 264 core biopsies (17%) were not given a score because of the lack of tumor cells. Seventy-eight percent of the individual discs on the microarray had scores in agreement with those of the whole-section slides. Among biopsy discs with tumor cells present, 94.5% were in agreement with the whole-section slide. The average probability that four randomly chosen biopsy discs, considered together, would accurately identify the presence of p53 staining in a whole section was 0.97 (95% CI.93-1.0). We conclude that tissue microarrays for squamous cell carcinomas can accurately represent immunohistochemical results of whole-slide specimens when four or more samples are used. Tissue microarrays are an important technique that may be applied to immunohistochemical studies of head and neck cancer.