Comprehensive genetic analysis of cancer cells

Human cancer is viewed as a disorder of genes originating from the progeny of a single cell that has accumulated multiple genetic alterations. The genetic alterations include point mutation, chromosomal rearrangements and imbalances. Amplifications primarily involve oncogenes whose overexpression leads to growth deregulation, while deletions commonly target tumor suppressor genes that control cell cycle checkpoints and DNA repair mechanisms. With the advent of molecular cytogenetics procedures for global detection of genomic imbalances and for multicolor visualization of structural chromosome changes, as well as the completion of human genome mapping and the development of microarray technology for serial gene expression analysis of the entire genomes, a significant progress has been made in uncovering the molecular basis of cancer. The major challenge in cancer biology is to decipher the molecular anatomy of various cancers and to identify cancer-related genes that now comprise only a fraction of human genes. The complete genetic anatomy of specific cancers would allow a better understanding of the role of genetic alterations in carcinogenesis, provide diagnostic and prognostic markers and discriminate between cells at different stages of progression toward malignancy. This review highlights current technologies that are available to explore cancer cells and outlines their application to investigations in human hepatocellular carcinoma.     

Multiparameter analysis and sorting of mammalian cells

An improved multisensor cell sorting instrument for quantitative analysis and sorting of cells has been developed. Cells stained with fluorescent dyes enter a flow chamber where cell volume, fluorescence, and light scatter sensors simultaneously measure multiple cellular properties. Cells then emerge in a liquid jet that is broken into uniform liquid droplets. Sensor signals are electronically processed in one of several ways for optimum cell discrimination and are displayed as pulse-amplitude distributions using a multichannel pulse-height analyzer. Processed signals activate cell sorting according to preselected parametric criteria by electrically charging droplets containing cells and electrostatically deflecting them into collection vessels. Illustrative examples of multiparameter cell analysis and sorting experiments using a model mouse tumor cell system, human and animal leukocytes, and cultured mammalian cells are presented.     

Cell sorting analysis of cell cycle-dependent X-ray sensitivity in end joining-deficient human cells

Non-homologous end joining (NHEJ) plays a major role in the repair of ionizing radiation-induced DNA double-strand breaks (DSBs), especially during the G1-phase of the cell cycle. Using a flow cytometric cell sorter, we fractionated G1- and S/G2-phase cells based on size to assess the DSB-repair activity in NHEJ factor-deficient DT40 and Nalm-6 cell lines. Colony formation assays revealed that the X-ray sensitivities of the G1-enriched populations correctly reflected the DSB-repair activities of both the DT40 and Nalm-6 cell lines. Furthermore, as assessed by γ-H2AX foci formation, the sorted cells exhibited less DNA damage than chemically synchronized cells. Given that it does not use fluorescent labeling or chemical agents, this method of cell sorting is simpler and less toxic than other methods, making it applicable to a variety of cell lines, including those that cannot be synchronized by standard chemical treatments.     

Exploiting differential effects of actomyosin contractility to control cell sorting among breast cancer cells

In order to gain a greater understanding of the factors that drive spatial organization in multicellular aggregates of cancer cells, we investigate the segregation patterns of 6 breast cell lines of varying degree of mesenchymal character during formation of mixed aggregates. Cell sorting is considered in the context of available adhesion proteins and cellular contractility. It is found that the primary compaction mediator (cadherins or integrins) for a given cell type in isolation plays an important role in compaction speed, which in turn is the major factor dictating preference for interior or exterior position within mixed aggregates. In particular, cadherin-deficient, invasion-competent cells tend to position towards the outside of aggregates, facilitating access to extracellular matrix. Reducing actomyosin contractility is found to have a differential effect on spheroid formation depending on compaction mechanism. Inhibition of contractility has a significant stabilizing effect on cell-cell adhesions in integrin-driven aggregation and a mildly destabilizing effect in cadherin-based aggregation. This differential response is exploited to statically control aggregate organization and dynamically rearrange cells in pre-formed aggregates. Sequestration of invasive cells in the interior of spheroids provides a physical barrier that reduces invasion in three-dimensional culture, revealing a potential strategy for containment of invasive cell types.     

Genome-wide detection and analysis of cell wall-bound proteins with LPxTG-like sorting motifs

Surface proteins of gram-positive bacteria often play a role in adherence of the bacteria to host tissue and are frequently required for virulence. A specific subgroup of extracellular proteins contains the cell wall-sorting motif LPxTG, which is the target for cleavage and covalent coupling to the peptidoglycan by enzymes called sortases. A comprehensive set of putative sortase substrates was identified by in silico analysis of 199 completely sequenced prokaryote genomes. A combination of detection methods was used, including secondary structure prediction, pattern recognition, sequence homology, and genome context information. With the hframe algorithm, putative substrates were identified that could not be detected by other methods due to errors in open reading frame calling, frameshifts, or sequencing errors. In total, 732 putative sortase substrates encoded in 49 prokaryote genomes were identified. We found striking species-specific variation for the LPxTG motif. A hidden Markov model (HMM) based on putative sortase substrates was created, which was subsequently used for the automatic detection of sortase substrates in recently completed genomes. A database was constructed, LPxTG-DB (http://bamics3.cmbi.kun.nl/sortase_substrates), containing for each genome a list of putative sortase substrates, sequence information of these substrates, the organism-specific HMMs based on the consensus sequence of the sortase recognition motif, and a graphic representation of this consensus.     

Autofluorescence flow sorting of breast cancer cell metabolism

Clinical cancer treatment aims to target all cell subpopulations within a tumor. Autofluorescence microscopy of the metabolic cofactors NAD(P)H and FAD has shown sensitivity to anti‐cancer treatment response. Alternatively, flow cytometry is attractive for high throughput analysis and flow sorting. This study measures cellular autofluorescence in three flow cytometry channels and applies cellular autofluorescence to sort a heterogeneous mixture of breast cancer cells into subpopulations enriched for each phenotype. Sorted cells were grown in culture and sorting was validated by morphology, autofluorescence microscopy, and receptor expression. Ultimately, this method could be applied to improve drug development and personalized treatment planning.

     

Fluorescence-activated single cell sorting of human embryonic stem cells

Human embryonic stem cells (hESC) are the subject of intense investigation for use in regenerative medicine, in toxicity testing, and as models for the study of human development. Automated cell sorting will enhance the isolation of homogenous pools of differentiated hESCs both for basic studies and for therapeutic applications. Sorting could also be used to deplete undifferentiated, potentially tumourigenic cells. However, hESCs are sensitive to single cell disaggregation and recover poorly when plated at clonal density. Here we report a method for successful semi-automated single cell sorting of hESCs. This method utilizes an ES-specific promoter-transgene construct and automated FACS-based single cell sorting and plating. Clonal recovery in physiologic oxygen (2%) was increased fourfold over room oxygen (21%; p < 0.01). This automated protocol will help to realize proposed hESC strategies that are hampered by low throughput and poor yields.     

Identification of a carboxylesterase expressed in protoplasts using fluorescence-activated cell sorting

A method was developed to identify plant carboxylesterases using a homologous expression system with the capacity for high-throughput screening based on fluorescence-activated cell sorting (FACS). Protoplasts of Arabidopsis thaliana were prepared and transfected with a mutated (Cys59Ser) Arabidopsis S-formylglutathione hydrolase ( atsfghm ), which encoded a carboxylesterase highly active in the hydrolysis of the vital marker methylumbelliferyl acetate (MUA) to the fluorophore methylumbelliferone (MU). Unlike all other Arabidopsis carboxylesterases studied to date, At SFGH and its more stable mutant variant At SFGHm are insensitive to inhibition by organophosphate insecticides, such as paraoxon. By making use of the combined traits of a high carboxylesterase activity towards MUA and a lack of sensitivity to paraoxon, FACS was employed to selectively collect catalytically active atsfghm -transformed protoplasts. A population of 400 000 protoplasts containing 8000 sfghm transformants was treated with paraoxon to inhibit endogenous esterase activity and then fed with MUA. Fluorescent cells expressing the At SFGHm enzyme were then collected by FACS, and the presence of the respective transgene was confirmed by polymerase chain reaction, with 9.6% of the transformants recovered. We suggest that the use of FACS to identify other carboxylesterases which can be catalytically determined using plant cell fluorescence-based assays could be a powerful method for the high-throughput screening of new enzymes, especially those which do not express well in microbial hosts.     

Anchored cell analysis and sorting (ACAS) system]

Advanced biology and recent technology have provided sophisticated and objective method for analyzing biological characteristics on cells. Following that, many new instruments have developed. Diagnostic immunocytochemistry has become an accepted diagnostic tool in cell biology. In recent years, remarkable advances in technology provide a method for quantitative and objective analyses of cell characteristics. The newly developed computer assisted laser cytometer (ACAS 570) can be applied in clinical basis as well as in research laboratory. Fluorescent intensities of ancharage-dependent cells can be automatically analyzed and make it possible to separate a subpopulation of cells. This computer controlled system principally consists of argon ion laser, phase contrast microscope. Quantitative fluorescence measurements and computer graphic images can be obtained. The present paper demonstrates multiple applications of laser cytometer for evaluation of cell biology.     

Optimizing optical flow cytometry for cell volume-based sorting and analysis

Cell size is a defining characteristic central to cell function and ultimately to tissue architecture. The ability to sort cell subpopulations of different sizes would facilitate investigation at genomic and proteomic levels of mechanisms by which cells attain and maintain their size. Currently available cell sorters, however, cannot directly measure cell volume electronically, and it would therefore be desirable to know which of the optical measurements that can be made in such instruments provide the best estimate of volume. We investigated several different light scattering and fluorescence measurements in several different cell lines, sorting cell fractions from the high and low end of distributions, and measuring volume electronically to determine which sorting strategy yielded the best separated volume distributions. Since we found that different optical measurements were optimal for different cell lines, we suggest that following this procedure will enable other investigators to optimize their own cell sorters for volume-based separation of the cell types with which they work.     

Identification of cancer stem cell markers in human malignant mesothelioma cells

Malignant mesothelioma (MM) is an aggressive and therapy-resistant neoplasm arising from the pleural mesothelial cells and usually associated with long-term asbestos exposure. Recent studies suggest that tumors contain cancer stem cells (CSCs) and their stem cell characteristics are thought to confer therapy-resistance. However, whether MM cell has any stem cell characteristics is not known. To understand the molecular basis of MM, we first performed serial transplantation of surgical samples into NOD/SCID mice and established new cell lines. Next, we performed marker analysis of the MM cell lines and found that many of them contain SP cells and expressed several putative CSC markers such as CD9, CD24, and CD26. Interestingly, expression of CD26 closely correlated with that of CD24 in some cases. Sorting and culture assay revealed that SP and CD24⁺ cells proliferated by asymmetric cell division-like manner. In addition, CD9⁺ and CD24⁺ cells have higher potential to generate spheroid colony than negative cells in the stem cell medium. Moreover, these marker-positive cells have clear tendency to generate larger tumors in mouse transplantation assay. Taken together, our data suggest that SP, CD9, CD24, and CD26 are CSC markers of MM and could be used as novel therapeutic targets.     

Purification of interstitial cells of Cajal by fluorescence-activated cell sorting

Interstitial cells of Cajal (ICC) in the gastrointestinal tract generate and propagate slow waves and mediate neuromuscular neurotransmission. Although damages to ICC have been described in several gastrointestinal motor disorders, analysis of their gene expression in health and disease has been problematic because of the difficulties in isolating these cells. Our goal was to develop techniques for large-scale purification of ICC. Murine ICC were identified in live gastrointestinal muscles with fluorescent Kit antibodies. Because this technique also labels resident macrophages nonspecifically, we attempted to separate ICC from these cells by fluorescence-activated cell sorting with or without immunomagnetic presorting. Efficacy and specificity of ICC purification were tested by quantitative RT-PCR of cell-specific markers. Fluorescence-based separation of small intestinal ICC from unlabeled cells and macrophages tagged with F4/80 antibodies yielded 30,000–40,000 cells and 60-fold enrichment of c-kit mRNA. However, the macrophage marker CD68 was also enriched 6-fold. Magnetic presorting of ICC did not significantly improve selectivity. After labeling contaminating cells with additional paramagnetic (anti-CD11b, -CD11c) and fluorescent antibodies (anti-CD11b) and depleting them by magnetic presorting, we harvested 2,000–4,000 cells from single gastric corpus-antrum muscles and detected an 30-fold increase in c-kit mRNA, no enrichment of mast cells, and an 4-fold reduction of CD68 expression. Adding labeled anti-CD45 antibody to our cocktail further increased c-kit enrichment and eliminated mast cells and macrophages. Smooth muscle cells and myenteric neurons were also depleted. We conclude that immunofluorescence-based sorting can yield ICC in sufficiently high numbers and purity to permit detailed molecular analyses. mouse; c-kit; macrophage; dendritic cell; mast cell     

Purification of transiently transfected cells by magnetic affinity cell sorting

A method was developed to purify transiently transfected HeLa cells or African green monkey kidney CV-1 cells by magnetic affinity cell sorting. Monolayer cultures were transfected with mammalian expression vectors coding for either of two novel cell surface antigens, the Tac subunit of the human IL-2 receptor or vesicular stomatitis virus G protein. During the transient expression phase, cell populations were placed in suspension and mixed with monoclonal-antibody-coated magnetic particles in the presence of a sorting solution designed to minimize nonspecific cell/cell and cell/particle interactions. Transfected cells expressing the vector-encoded cell surface antigen were then isolated by application of a magnetic field. Reconstruction experiments indicated that IL-2 receptor-positive cells were bound about 100-fold more efficiently than receptor-negative cells. In transient transfection experiments, populations of greater than 90% antigen-positive cells were reproducibly obtained.     

Isolation of glucocorticoid-unresponsive rat hepatoma cells by fluorescence-activated cell sorting

We have used a mouse mammary tumor virus (MMTV)-infected rat hepatoma cell line as a model system for studying glucocorticoid action. These cells induce tyrosine aminotransferase and MMTV in response to the synthetic glucocorticoid, dexamethasone. The major viral antigen, a glycoprotein of 52,000 daltons (gp52), appears on the surface of infected cells in amounts which reflect the cytoplasmic content of viral RNA. Using an anti-gp52 antiserum and a fluorescence-activated cell sorter (FACS), we have selected variants which display low levels of pg52 in the presence of the hormone. Multiple cycles of enrichment for cells that fluoresce weakly in the presence of hormone have generated a population which fails to produce a detectable increase in cell surface gp52 in response to dexamethasone. This population of nonresponders and a number of independent clones derived from this population were analyzed for their ability to induce gp52 and TAT and for these presence of glucocorticoid receptors. All nonresponder clones exhibited little or no induction of either glucocorticoid-inducible marker. Two of the clones contained reduced levels of glucocrticoid receptor, while the remainder of the clones showed no detectable specific hormone binding. These results provide genetic evidence that a single class of glucocorticoid receptors is involved in the induction of both MMTV and TAT in HTC cells.     

Genomic and kinetic analysis of novel Nitrospinae enriched by cell sorting

Chemolithoautotrophic nitrite-oxidizing bacteria (NOB) are key players in global nitrogen and carbon cycling. Members of the phylum Nitrospinae are the most abundant, known NOB in the oceans. To date, only two closely affiliated Nitrospinae species have been isolated, which are only distantly related to the environmentally abundant uncultured Nitrospinae clades. Here, we applied live cell sorting, activity screening, and subcultivation on marine nitrite-oxidizing enrichments to obtain novel marine Nitrospinae. Two binary cultures were obtained, each containing one Nitrospinae strain and one alphaproteobacterial heterotroph. The Nitrospinae strains represent two new genera, and one strain is more closely related to environmentally abundant Nitrospinae than previously cultured NOB. With an apparent half-saturation constant of 8.7 ± 2.5 µM, this strain has the highest affinity for nitrite among characterized marine NOB, while the other strain (16.2 ± 1.6 µM) and Nitrospina gracilis (20.1 ± 2.1 µM) displayed slightly lower nitrite affinities. The new strains and N. gracilis share core metabolic pathways for nitrite oxidation and CO₂ fixation but differ remarkably in their genomic repertoires of terminal oxidases, use of organic N sources, alternative energy metabolisms, osmotic stress and phage defense. The new strains, tentatively named “Candidatus Nitrohelix vancouverensis” and “Candidatus Nitronauta litoralis”, shed light on the niche differentiation and potential ecological roles of Nitrospinae.Subject terms: Water microbiology, Microbial ecology, Biogeochemistry