Clinical applications of laser scanning cytometry

This study reviews existing and potential clinical applications of laser scanning cytometry (LSC) and outlines possible future developments. LSC provides a technology for solid phase cytometry. Fluorochrome-labeled specimens are immobilized on microscopic slides that are placed on a conventional epifluorescence microscope and analyzed by one or two lasers. Data comparable to flow cytometry are generated. In addition, the position of each event is recorded, a feature that allows relocalization and visualization of each measured event. The major advantage of LSC compared with other cytometric methods is the combination of two features: (a) the minimal clinical sample volume needed and (b) the connection of fluorescence data and morphological information for the measured event. Since the introduction of LSC, numerous methods have been established for the analysis of cells, cellular compartments, and tissues. Although most cytometric methods use only two or three colors, the characterization of specimens with up to five fluorochromes is possible. Most clinical applications have been designed to determine ploidy and immunophenotype; other applications include analyses of tissue biopsies and sections, fluorescence in situ hybridization, and the combination of vital and nonvital information on a single-cell basis. With the currently available assays, LSC has proven its wide spectrum of clinical applicability in slide-based cytometry and can be introduced as a standard technology in multiple clinical settings.     

Circulating tumour cells lacking cytokeratin in breast cancer: the importance of being mesenchymal

Circulating tumour cells (CTCs) are independent predictor of prognosis in metastatic breast cancer. Nevertheless, in one third of patients, circulating tumour cells are undetected by conventional methods. Aim of the study was to assess the prognostic value of circulating tumour cells expressing mesenchymal markers in metastatic breast cancer patients. We isolated CTC from blood of 55 metastatic breast cancer patients. CTC were characterized for cytokeratins and markers of epithelial mesenchymal transition. The gain of mesenchymal markers in CTC was correlated to prognosis of patients in a follow-up of 24 months. The presence of mesenchymal markers on CTC more accurately predicted worse prognosis than the expression of cytokeratins alone. Because of the frequent loss of epithelial antigens by CTC, assays targeting epithelial antigens may miss the most invasive cell population. Thus, there is an urgent need to improve detection methods to identify CTC which undergone epithelial mesenchymal transition program.     

Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or F?rster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs.     

Use of chlortetracycline fluorescence for the detection of Ca storing intracellular vesicles in normal human erythrocytes

The uptake of chlortetracycline (CTC) and the nature of the fluorescence of CTC was studied in intact human erythrocytes from apparently healthy donors. The uptake of CTC at 22 degrees C proceeded with a t1/2 of about 3 min, and after 15 min a stable equilibrium was achieved with an intracellular accumulation by a factor of 5-6 relative to the medium concentration. The accumulation did not change in the range of CTC concentrations tested (20-500 microM). The Ca specificity of the CTC fluorescence spectrum was confirmed by Ca depletion of red cells using A23187 in the presence of EGTA and 0.2 mM Mg. This procedure decreased the total intracellular calcium content by about 70% and reduced the fluorescence intensity to one-fourth. Fluorescence microscopy of red cells incubated with 100 microM CTC at 22 degrees C showed that the fluorescence originated mainly from the red cell membrane. In addition, in about 15% of erythrocytes one or more fluorescent dots (diameter greater than 0.2 less than 1 microns) were detected. The fluorescence of the dots and membranes was related to calcium, as evidenced by the reduction of their intensity in Ca depleted cells. The number of erythrocytes with fluorescent dots and the frequency of the dots per cell was largely unaffected by lowering the incubation temperature to 0 degrees C, indicating that the dots most probably do not represent endocytotic artifacts induced by CTC. The number of dots was increased in erythrocytes preincubated with primaquine, demonstrating that CTC fluorescence can be applied to monitor the appearance of intracellular Ca storing vesicles. It is concluded that in (at least) 15% of erythrocytes obtained from apparently healthy donors intracellular vesicles containing Ca can be detected by CTC fluorescence microscopy.     

Utilizing circulating tumor cells: challenges and pitfalls

Circulating tumor cells (CTC) detected in the blood of cancer patients could be used for risk-stratification, molecular subclassification and as an intermediate end-point in therapeutic efficacy studies. Most studies to date have focused on enumeration of CTC in advanced cancer patients but further development of CTC evaluation technologies could allow expansion into early disease, monitoring of treatment response, and selection of patients for targeted therapies based on a CTC derived signature. This review discusses the challenges faced in achieving these goals, including the potential absence of CTC in patients with no blood-borne metastases, CTC intra-patient molecular heterogeneity, ex vivo loss of CTC viability, and the biological differences between CTC and metastatic tissue.     

Culture of Drosophila S2 cells and their use for RNAi-mediated loss-of-function studies and immunofluorescence microscopy

Cultured Drosophila cell lines have become an increasingly popular model system for cell biological and functional genomic studies. One of the most commonly used lines, S2 cells, is particularly useful as it is easy to grow and maintain in the lab, is highly susceptible to gene inhibition using RNAi and is well suited to high-resolution light microscopic assays. Here, we provide protocols for the routine culture and RNAi treatment of S2 cells and methods to prepare these cells for fluorescence microscopy. Using these techniques, loss-of-function experiments may be performed after 4-7 d of RNAi-mediated protein depletion.     

A prognostic gene expression profile that predicts circulating tumor cell presence in breast cancer patients

The detection of circulating tumor cells (CTCs) in the peripheral blood and microarray gene expression profiling of the primary tumor are two promising new technologies able to provide valuable prognostic data for patients with breast cancer. Meta-analyses of several established prognostic breast cancer gene expression profiles in large patient cohorts have demonstrated that despite sharing few genes, their delineation of patients into "good prognosis" or "poor prognosis" are frequently very highly correlated, and combining prognostic profiles does not increase prognostic power. In the current study, we aimed to develop a novel profile which provided independent prognostic data by building a signature predictive of CTC status rather than outcome. Microarray gene expression data from an initial training cohort of 72 breast cancer patients for which CTC status had been determined in a previous study using a multimarker QPCR-based assay was used to develop a CTC-predictive profile. The generated profile was validated in two independent datasets of 49 and 123 patients and confirmed to be both predictive of CTC status, and independently prognostic. Importantly, the "CTC profile" also provided prognostic information independent of the well-established and powerful '70-gene' prognostic breast cancer signature. This profile therefore has the potential to not only add prognostic information to currently-available microarray tests but in some circumstances even replace blood-based prognostic CTC tests at time of diagnosis for those patients already undergoing testing by multigene assays.     

Visualization of single fluorophores in living cells

Methods applicable to visualizing single fluorophores in living cells are described, namely, laser epifluorescence, confocal, near-field, two-photon, and total internal reflection microscopy. It is demonstrated that total internal reflection microscopy is the most appropriate for visualizing single fluorophores near the substrate-medium interface. This method can be used for studying receptors, ion channels, and numerous cytoskeletal and signal molecules located on or near the basal cell membrane. It is demonstrated that stringent criteria are necessary when identifying single molecules, as these objects emit a limited number of photons before irreversible photobleaching and their fluorescence is obscured by autofluorescence or out-of-focus fluorescence. The methods used for studying the lateral mobility of single molecules floating on the cell membrane are also described.     

Circulating tumour cells in clinical practice: Methods of detection and possible characterization

Circulating Tumour Cells (CTCs) can be released from the primary tumour into the bloodstream and may colonize distant organs giving rise to metastasis. The presence of CTCs in the blood has been documented more than a century ago, and in the meanwhile various methods have been described for their detection. Most of them require an initial enrichment step, since CTCs are a very rare event. The different technologies and also the differences among the screened populations make the clinical significance of CTCs detection difficult to interprete. Here we will review the different assays up to now available for CTC detection and analysis. Moreover, we will focus on the relevance of the clinical data, generated so far and based on the CTCs analysis. Since the vast majority of data have been produced in breast cancer patients, the review will focus especially on this malignancy.     

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. II. Subcellular localization of the fluorescent probe chlorotetracycline

Subcellular distribution of the divalent cation-sensitive probe chlorotetracycline (CTC) was observed by fluorescence microscopy in isolated pancreatic acinar cells, dissociated hepatocytes, rod photoreceptors, and erythrocytes. In each cell type, areas containing membranes fluoresced intensely while areas containing no membranes (nuclei and zymogen granules) were not fluorescent. Cell compartments packed with rough endoplasmic reticulum or Golgi vesicles (acinar cells) or plasma membrane-derived membranes (rod outer segments) exhibited a uniform fluorescence. In contrast, cell compartments having large numbers of mitochondria (hepatocytes and the rod inner segment) exhibited a punctate fluorescence. Punctate fluorescence was prominent in the perinuclear and peri-granular areas of isolated acinar cells during CTC efflux, suggesting that under these conditions mitochondrial fluorescence may account for a large portion of acinar cell fluorescence. Fluorometry of dissociated pancreatic acini, preloaded with CTC, showed that application of the mitochondrial inhibitors antimycin A, NaCN, rotenone, or C1CCP, or of the divalent cation ionophore A23187 (all agents known to release mitochondrial calcium) rapidly decreased the fluorescence of acini. In the case of mitochondrial inhibitors, this response could be elicited before but not following the loss of CTC fluorescence induced by bethanechol stimulation. Removal of extracellular Ca2+ and Mg2+ or addition of EDTA also decreased fluorescence but did not prevent secretagogues or mitochondrial inhibitors from eliciting a further response. These data suggest that bethanechol acts to decrease CTC fluorescence at the same intracellular site as do mitochondrial inhibitors. This could be due to release of calcium from either mitochondria or another organelle that requires ATP to sequester calcium.     

Comparative reliability and sensitivity of different methods for assessing treatment effects on sperm production

Investigators may choose among a variety of different approaches for identifying and quantifying potential treatment effects on sperm production rates. Should a treatment actually alter sperm production, the likelihood that the response will be detected and declared statistically significant depends on several factors. These include the size of the treatment response, the inherent variability associated with the endpoint of interest among normal, untreated males, and the number of replicate animals per treatment group. This study was undertaken to characterize the relative power and sensitivity of several widely used methods for quantifying sperm production, for the purpose of identifying those that would be the most likely to enable detection of actual treatment effects. For this exercise, the typical inherent variability associated with several endpoints of testicular function in rats, rabbits and humans was established by a review of the published literature. Based on this typical variability and a components-of-variance approach, the relative number of replicates needed to provide experiments of equivalent power and sensitivity was determined. Consideration was also given to the likely nature of changes in the testis in response to adverse treatments. Based on all of this information, the methods for quantifying spermatogenesis that would be most likely to detect an actual treatment effect were determined to be as follows (from greatest to least powerful and sensitive): (1) quantification of round spermatids per Sertoli cell or per seminiferous tubular cross-section, (2) the determination of the number of spermatids or daily sperm production (DSP) per gram of testis or per testis via volume density approaches, (3) the determination of the number of elongated spermatids or DSP per gram or per testis via the testicular homogenate approach, and (4) the direct enumeration of degenerating germ cells.     

Circulating tumor cell isolation,culture, and downstream molecular analysis

Circulating tumor cells (CTCs) are a major contributor of cancer metastases and hold a promising prognostic significance in cancer detection. Performing functional and molecular characterization of CTCs provides an in-depth knowledge about this lethal disease. Researchers are making efforts to design devices and develop assays for enumeration of CTCs with a high capture and detection efficiency from whole blood of cancer patients. The existing and on-going research on CTC isolation methods has revealed cell characteristics which are helpful in cancer monitoring and designing of targeted cancer treatments. In this review paper, a brief summary of existing CTC isolation methods is presented. We also discuss methods of detaching CTC from functionalized surfaces (functional assays/devices) and their further use for ex-vivo culturing that aid in studies regarding molecular properties that encourage metastatic seeding. In the clinical applications section, we discuss a number of cases that CTCs can play a key role for monitoring metastases, drug treatment response, and heterogeneity profiling regarding biomarkers and gene expression studies that bring treatment design further towards personalized medicine.     

Optical coding of mammalian cells using semiconductor quantum dots

Cell-based assays are widely used to screen compounds and study complex phenotypes. Few methods exist, however, for multiplexing cellular assays or labeling individual cells in a mixed cell population. We developed a generic encoding method for cells that is based on peptide-mediated delivery of quantum dots (QDs) into live cells. The QDs are nontoxic and photostable and can be imaged using conventional fluorescence microscopy or flow cytometry systems. We created unique fluorescent codes for a variety of mammalian cell types and show that our encoding method has the potential to create > 100 codes. We demonstrate that QD cell codes are compatible with most types of compound screening assays including immunostaining, competition binding, reporter gene, receptor internalization, and intracellular calcium release. A multiplexed calcium assay for G-protein-coupled receptors using QDs is demonstrated. The ability to spectrally encode individual cells with unique fluorescent barcodes should open new opportunities in multiplexed assay development and greatly facilitate the study of cell/cell interactions and other complex phenotypes in mixed cell populations.     

An electrical biosensor for the detection of circulating tumor cells

In this report, we demonstrate a semi-integrated electrical biosensor for the detection of rare circulating tumor cells (CTCs) in blood. The sample was first enriched through a combination of immunomagnetic isolation and size filtration. The integration of both methods provided a high enrichment performance with a recovery rate above 70%, even for very low numbers of cancer cells present in the original sample (10 spiked MCF7 cells in 0.5 mL of blood). In the same system, the sample was then transferred to a microchip for further magnetic concentration, followed by immunochemical trapping and electronic detection by impedance spectroscopy. Three levels of spiked CTC number (30±2, 124±29, 273±23) in 10 μL of filtered blood sample were distinguished by monitoring the impedance change of the microelectrode array (MEA). The integration of different functions in a single system provided a methodology to process milliliter-sized blood samples at the macroscale and interface with the microdimensions of a highly sensitive electronic detector. The results showed that the whole system was able to detect different levels of spiked cancer cells without the use of time- and cost-intensive fluorescence labeling and image analysis. This has the potential to provide clinicians with a standalone system to monitor changes in CTC numbers throughout therapy conveniently and frequently for efficient cancer treatments.     

Circulating tumor cells: highlight on practical implications

Circulating tumor cells (CTCs) are cells of presumed epithelial origin, whose prognostic and predictive value in metastatic cancer patients has recently been demonstrated. To date, the count of CTCs through the CellSearch? system represents a valid approach for monitoring disease status in patients with metastatic colorectal, breast, and prostate cancer; in these cancer types, a rise in the CTC count at any time during treatment predicts a poor outcome. Nevertheless, the clinical utility of monitoring CTC counts remains controversial, and what to do when CTC counts rise during therapy still remains an unanswered question. In this report, we suggest how to integrate CTC counts with their molecular characterization to better translate biologic information obtained on CTCs into daily clinical practice.     

Comparison of methods for measuring oxygen consumption in tumor cells in vitro

The oxygen consumption rate of tumor cells affects tumor oxygenation and response to therapies. Highly sensitive methods for determining cellular oxygen consumption are, therefore, needed to identify treatments that can modulate this parameter. We compared the performances of three different methods for measuring cellular oxygen consumption: electron paramagnetic resonance (EPR) oximetry, the Clark electrode, and the MitoXpress fluorescent assay. To compare the assays, we used K562 cells in the presence of rotenone and hydrocortisone, compounds that are known to inhibit the mitochondrial electron transport chain to different extents. The EPR method was the only one that could identify both rotenone and hydrocortisone as inhibitors of tumor cell oxygen consumption. The Clark electrode and the fluorescence assay demonstrated a significant decrease in cellular oxygen consumption after administration of the most potent inhibitor (rotenone) but failed to show any significant effect of hydrocortisone. EPR oximetry is, therefore, the most sensitive method for identifying inhibitors of oxygen consumption on cell assays, whereas the Clark electrode offers the unique opportunity to add external compounds during experiments and still shows great sensitivity in studying enzyme and chemical reactions that consume oxygen (non-cell assays). Finally, the MitoXpress fluorescent assay has the advantage of a high-sample throughput and low bulk requirements but at the cost of a lower sensitivity.     

Apoptosis of circulating tumor cells in prostate cancer patients.

BACKGROUND: The prescence of circulating tumor cells (CTCs) in the peripheral blood of cancer patients and their frequency has been correlated with disease status. METHODS: In this study, CTCs were characterized by flow cytometry and fluorescence microscopy after immunomagnetic enrichment from 7.5-ml blood samples collected from patients with prostate cancer in evacuated blood-draw tubes that contained an anticoagulant and a preservative. Events were classified as tumor cell candidates if they expressed cytokeratin, lacked CD45, and stained with the nucleic acid dye 4,6-diamidino-2-phenylindole. RESULTS: In the blood of prostate cancer patients, only few of these events were intact cells. Other CTC events appeared as damaged cells or cell fragments by microscopy. By flow cytometry, these events stained variably with 4,6-diamidino-2-phenylindole and frequently expressed the apoptosis-induced, caspase-cleaved cytokeratin 18. Similar patterns of cell disintegration were observed when cells of the prostate line LNCaP were exposed to paclitaxel before spiking the cells into normal blood samples. CONCLUSIONS: The different observed stages of tumor cell degradation or apoptosis varied greatly between patients and were not found in blood of normal donors. Enumeration of CTCs and identification of CTCs undergoing apoptosis may provide relevant information to evaluate the response to therapy in cancer patients.     

Circulating tumor cells: detection,molecular profiling and future prospects

Disseminated malignancy is responsible for the vast majority of cancer-related deaths. During this process, circulating tumor cells (CTC) are generated, spread from the primary tumor, colonize distant organs and lead to overt metastatic disease. CTC are essential for establishing metastasis; however, they are not sufficient as this process is highly inefficient and most will fail to grow in target sites. Several CTC die during migration while others remain dormant for several years and very few grow into macrometastases. CTC have been well documented in the bloodstream of cancer patients; however, the clinical relevance of this detection is still the subject of controversies and their biology is poorly understood. Indeed, available markers fail to distinguish between subgroups of CTC, and several current methods lack sensitivity, specificity or reproducibility in CTC characterization and detection. The advent of more precise technologies is renewing the interest in CTC biology. We will review herein recent findings on CTC biology, on the role of host–tumor interactions in CTC shedding and implantation, available methods of CTC detection and future perspectives for the molecular characterization of the CTC subset(s) responsible for the development of metastasis. Ultimately, understanding CTC biology and host–tumor ‘complementarities’ will help define metastasis-related biomarkers providing formidable and tailored novel therapeutic targets.     

Circulating tumor cells: detection, molecular profiling and future prospects

Disseminated malignancy is responsible for the vast majority of cancer-related deaths. During this process, circulating tumor cells (CTC) are generated, spread from the primary tumor, colonize distant organs and lead to overt metastatic disease. CTC are essential for establishing metastasis; however, they are not sufficient as this process is highly inefficient and most will fail to grow in target sites. Several CTC die during migration while others remain dormant for several years and very few grow into macrometastases. CTC have been well documented in the bloodstream of cancer patients; however, the clinical relevance of this detection is still the subject of controversies and their biology is poorly understood. Indeed, available markers fail to distinguish between subgroups of CTC, and several current methods lack sensitivity, specificity or reproducibility in CTC characterization and detection. The advent of more precise technologies is renewing the interest in CTC biology. We will review herein recent findings on CTC biology, on the role of host-tumor interactions in CTC shedding and implantation, available methods of CTC detection and future perspectives for the molecular characterization of the CTC subset(s) responsible for the development of metastasis. Ultimately, understanding CTC biology and host-tumor 'complementarities' will help define metastasis-related biomarkers providing formidable and tailored novel therapeutic targets.