Detection and quantification of circulating tumor cells in mouse models of human breast cancer using immunomagnetic enrichment and multiparameter flow cytometry.

BACKGROUND: Circulating tumor cells (CTCs) in the peripheral blood of breast cancer patients may be an important indicator of metastatic disease and poor prognosis. However, the use of experimental models is required to fully elucidate the functional consequences of CTCs. The purpose of this study was to optimize the sensitivity of multiparameter flow cytometry for detection of human tumor cells in mouse models of breast cancer. METHODS: MDA-MB-468 human breast cancer cells were serially diluted in whole mouse blood. Samples were lysed and incubated with a fluorescein isothiocyanate-conjugated anti-human leukocytic antigen antibody and a phycoerythrin-conjugated anti-mouse pan-leukocyte CD45 antibody. Samples were then immunomagnetically depleted of CD45-positive leukocytes, fixed, permeabilized, and stained with propidium iodide before flow cytometric analysis. RESULTS: Human breast cancer cells could be differentiated from mouse leukocytes based on increased light scatter, cell surface marker expression, and aneuploid DNA content. The method was found to have a lower sensitivity limit of 10(-5) and was effective for detecting human breast cancer cells in vivo in the circulation of experimental mice carrying primary human mammary tumors. CONCLUSIONS: This technique has the potential to be a valuable and sensitive tool for investigating the biological relevance of CTCs in experimental mouse models of breast cancer.     

A fluorescence microplate assay using yopro-1 to measure apoptosis: application to HL60 cells subjected to oxidative stress

A new one-step labeling procedure using the membrane permeant fluorescent probe yopro-1 in association with fluorescence microtitration for the rapid determination of apoptosis is reported. Programmed cell death was induced by the pro-apoptotic agents etoposide and staurosporine, and measured in nonadherent HL60 cells and adherent phorbol 12-myristate 13-acetate (PMA)-treated HL60 cells. Cell viability was controlled by trypan blue exclusion and calcein-AM staining. To confirm results of fluorescence microplate assay, apoptosis was measured by flow cytometry analysis using the same fluorescent probe, and results showed corresponding data between both procedures. Development of apoptosis was confirmed by the presence of PARP (poly(ADP-ribose) polymerase cleavage and nuclear DAPI (4,6-diamidino-2-phenylindole) staining, two well-known methods used to investigate apoptosis. The fluorescence microplate assay was also applied to measure apoptosis in cells exposed to an oxidative stress induced by tert-butylhydroperoxide (t-BHP), and results confirmed the potential of the fluorescence microplate assay in measuring events of apoptosis, especially in adherent, cultured, living cells.     

Double-fluorescence image microscopy for quantitation of prostate-specific antigen in histologic sections of the prostate

BACKGROUND: Although the assessment of serum prostate-specific antigen (PSA) has become a powerful instrument in the diagnosis and for prognosis of prostate carcinoma, there are few quantitative studies of PSA in tissue sections. METHODS: We developed a technique using double-fluorescence image microscopy for quantifying immunohistochemical reactions in tissue sections. PSA was stained by Texas Red and the cellular DNA was counterstained with 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI). The fluorescence of Texas Red and DAPI was quantified separately after subtraction of background and shading correction. The amount of PSA related to the amount of DNA in identical tissue parts was studied in archival specimens from patients with hyperplasia and prostate carcinoma. RESULTS: The amount of tissue PSA decreased with the increase in tumor grade, Gleason score, and the change from diploid to aneuploid. CONCLUSION: Double-fluorescence image microscopy is a valuable technique for obtaining quantitative information of cellular constituents. For standardization of immunochemical reactions in tissue sections, cellular DNA seems to be most appropriate.     

Optimization and Evaluation of a Novel Size Based Circulating Tumor Cell Isolation System

Isolation of circulating tumor cells (CTCs) from peripheral blood has the potential to provide a far easier “liquid biopsy” than tumor tissue biopsies, to monitor tumor cell populations during disease progression and in response to therapies. Many CTC isolation technologies have been developed. We optimized the Parsortix system, an epitope independent, size and compressibility-based platform for CTCs isolation, making it possible to harvest CTCs at the speed and sample volume comparable to standard CellSearch system. We captured more than half of cancer cells from different cancer cell lines spiked in blood samples from healthy donors using this system. Cell loss during immunostaining of cells transferred and fixed on the slides is a major problem for analyzing rare cell samples. We developed a novel cell transfer and fixation method to retain >90% of cells on the slide after the immunofluorescence process without affecting signal strength and specificity. Using this optimized method, we evaluated the Parsortix system for CTC harvest in prostate cancer patients in comparison to immunobead based CTC isolation systems IsoFlux and CellSearch. We harvested a similar number (p = 0.33) of cytokeratin (CK) positive CTCs using Parsortix and IsoFlux from 7.5 mL blood samples of 10 prostate cancer patients (an average of 33.8 and 37.6 respectively). The purity of the CTCs harvested by Parsortix at 3.1% was significantly higher than IsoFlux at 1.0% (p = 0.02). Parsortix harvested significantly more CK positive CTCs than CellSearch (p = 0.04) in seven prostate cancer patient samples, where both systems were utilized (an average of 32.1 and 10.1 respectively). We also captured CTC clusters using Parsortix. Using four-color immunofluorescence we found that 85.8% of PC3 cells expressed EpCAM, 91.7% expressed CK and 2.5% cells lacked both epithelial markers. Interestingly, 95.6% of PC3 cells expressed Vimentin, including those cells that lacked both epithelial marker expression, indicating epithelial-to-mesenchymal transition. CK-positive/Vimentin-positive/CD45-negative, and CK-negative/Vimentin-positive/CD45-negative cells were also observed in four of five prostate cancer patients but rarely in three healthy controls, indicating that Parsortix harvests CTCs with both epithelial and mesenchymal features. We also demonstrated using PC3 and DU145 spiking experiment that Parsortix harvested cells were viable for cell culture.     

A rare phenotype of phosphoglucomutase-2 first detected in Mongoloids

Summary Improvements in the accuracy of DNA content determination by flow cytometry might allow the detection of aneuploidy in interphase cells to serve as an adjunct to conventional cytogenetics. We compared alternate methods of staining and fluorescence standardization in analyses of human diploid and constitutionally aneuploid cultured fibroblast-like cells. Optimum results were obtained using the dye DAPI (4,6-diamidino-2-phenylindole) and an avian red blood cell standard. The standard error of the estimate of DNA content by this technique was 0.7%–0.96%, approaching more closely than previously the range necessary for reliable discrimination of numerical chromosomal aberrations.Supported by NIH grants GM-15253, AG-01751 and DFG Grant SFB 105     

Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

Hematogenous metastasis accounts for the majority of cancer-related deaths, yet the mechanism remains unclear. Circulating tumor cells (CTCs) in blood may employ different pathways to cross blood endothelial barrier and establish a metastatic niche. Several studies provide evidence that prostate cancer (PCa) cell tethering and rolling on microvascular endothelium via E-selectin/E-selectin ligand interactions under shear flow theoretically promote extravasation and contribute to the development of metastases. However, it is unknown if CTCs from PCa patients interact with E-selectin expressed on endothelium, initiating a route for tumor metastases. Here we report that CTCs derived from PCa patients showed interactions with E-selectin and E-selectin expressing endothelial cells. To examine E-selectin-mediated interactions of PCa cell lines and CTCs derived from metastatic PCa patients, we used fluorescently-labeled anti-prostate specific membrane antigen (PSMA) monoclonal antibody J591-488 which is internalized following cell-surface binding. We employed a microscale flow device consisting of E-selectin-coated microtubes and human umbilical vein endothelial cells (HUVECs) on parallel-plate flow chamber simulating vascular endothelium. We observed that J591-488 did not significantly alter the rolling behavior in PCa cells at shear stresses below 3 dyn/cm². CTCs obtained from 31 PCa patient samples showed that CTCs tether and stably interact with E-selectin and E-selectin expressing HUVECs at physiological shear stress. Interestingly, samples collected during disease progression demonstrated significantly more CTC/E-selectin interactions than samples during times of therapeutic response (p=0.016). Analysis of the expression of sialyl Lewis X (sLe^x) in patient samples showed that a small subset comprising 1.9-18.8% of CTCs possess high sLe^x expression. Furthermore, E-selectin-mediated interactions between prostate CTCs and HUVECs were diminished in the presence of anti-E-selectin neutralizing antibody. CTC-Endothelial interactions provide a novel insight into potential adhesive mechanisms of prostate CTCs as a means to initiate metastasis.     

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche. The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines. Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions. Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs. Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions. However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly. CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs. Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques. In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system. This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.     

A simple multicolor flow cytometry protocol for detection and molecular characterization of circulating tumor cells in epithelial cancers

Circulating tumor cells (CTCs) might not only serve as prognostic marker but could also be useful for monitoring treatment efficacy. A multicolor flow cytometry protocol for their detection and molecular characterization in peripheral blood was developed which consisted of erythrocyte lysis followed by staining of cells with fluorochrome-labeled antibodies against CD45 and the epithelial markers EpCam and cytokeratin 7/8. For reducing the number of events acquired by flow cytometry, an electronic threshold for the fluorescent signals from the epithelial markers was applied. After establishment of the protocol by using spiking experiments, its suitability to determine the absolute number of CTCs as well as their expression of epidermal growth factor receptor (EGFR) and its phosphorylated form (phospho-EGFR) in blood samples from patients with squamous cell carcinoma of the head and neck (SCCHN) was validated. Spiking experiments demonstrated an excellent recovery (mean 85%) and a linear performance (R(2) = 0.98) of the protocol. Sensitivity and specificity were comparable to our former protocol using immunomagnetic CTC pre-enrichment. The analysis of 33 SCCHN patient samples revealed the presence of CTCs in 33.3% of cases with a mean ± SD of 1.5 ± 0.5 CTCs per 3.75 ml blood. EGFR was expressed in 100% and phospho-EGFR in 36.4% of the CTC+ cases. We have established a simple and sensitive multicolor flow cytometry protocol for detection of CTCs in patients with epithelial cancers including SCCHN which will allow their detailed molecular characterization.     

Extracorporeal Photo-Immunotherapy for Circulating Tumor Cells

It is well established that metastasis through the circulatory system is primarily caused by circulating tumor cells (CTCs). In this preliminary effort, we report an approach to eliminate circulating tumor cells from the blood stream by flowing the blood though an extracorporeal tube and applying photodynamic therapy (PDT). Chlorin e6 (Ce6), a photosensitizer, was conjugated to CD44 antibody in order to target PC-3, a prostate cancer cell line. PC-3 cells were successfully stained by the Ce6-CD44 antibody conjugate. PDT was performed on whole blood spiked with stained PC-3 cells. As the blood circulated through a thin transparent medical tube, it was exposed to light of 660 nm wavelength generated by an LED array. An exposure of two minutes was sufficient to achieve selective cancer cell necrosis. In comparison, to PDT of cells growing inside a tissue culture, the PDT on thin tube exhibited significantly enhanced efficiency in cell killing, by minimizing light attenuation by blood. It suggests a new extracorporeal methodology of PDT for treating CTCs as well as other hematological pathogens.     

Assessment of cell viability by flow cytometric analysis using DNase exclusion

A new method was developed for selective measurement of DNA distributions in viable cell populations. The method is based on the fact that non-viable cells lose membrane integrity and treatment of such cells with DNase should remove their DNA. The DNase-treated cells were stained with DNA fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) in the presence of Triton X-100. DNA distribution was measured by flow cytometry prior to and after treatment with DNase. Percentage of cells stained after DNase treatment was considered as an index of cell viability. Optimal conditions for DNase treatment and application of DNase exclusion test for the analysis of spontaneous cell death, selective death of cells arrested in S/G2 phases, instant cell disintegration induced by cytotoxic compounds and cell death induced by hyperthermia are described.     

Validation of a multiparameter flow cytometry method for the determination of phosphorylated extracellular-signal-regulated kinase and DNA in circulating tumor cells

A simple, selective, and sensitive multiparameter fluorescence activated cell sorting method utilizing density gradient centrifugation and magnetic antibody cell sorting was developed and validated for the determination of phosphorylated extracellular-signal-regulated kinase (pERK) and DNA in circulating tumor cells (CTCs). Cell preparation tubes (CPT) were used for peripheral blood collection and density gradient centrifugation, followed by phosphorylation of ERK with epidermal growth factor (EGF). After fixation with formaldehyde and methanol, magnetic anti epithelial cell adhesion molecule (EpCAM) micro-beads were used for the selective isolation of CTCs from the background, consisting of peripheral blood mononuclear cells and platelets. Subsequently, samples were stained with Hoechst 33342, and fluorescent antibodies against EpCAM, CD45, and pERK. Flow cytometry was used for identification and enumeration of CTCs and determination of their pERK and DNA content. The validation parameters included specificity, recovery, linearity, precision, sensitivity, and stability. The lower limit of quantification was two CTCs per 8 ml peripheral blood. Samples were stable for 4 months in storage at -80°C. The applicability of the method was demonstrated by successful enumeration of CTCs, and the determination of DNA, and pERK before and after stimulation with EGF in 8 ml peripheral blood samples from patients with metastatic cancer.     

Early cellular events during induction of carrot explants with 2,4-D

Summary The cellular events occurring in carrot hypocotyl explants during long-term and pulse treatment with 2,4-D were followed using different techniques (light and transmission electron microscopy, flow cytometry, PCNA staining). Different morphogenetic pathways were induced under the various experimental conditions. Nevertheless, in the explants the activated cells were the same (provascular cells) and they showed very similar structural and ultrastructural changes. The long-term treatment with 2,4-D induced rapid re-activation of the cell cycle.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - PCNA proliferating cell nuclear antigen - ER endoplasmic reticulum - GA Golgi apparatus     

Morphological Differences between Circulating Tumor Cells from Prostate Cancer Patients and Cultured Prostate Cancer Cells

Circulating tumor cell (CTC) enumeration promises to be an important predictor of clinical outcome for a range of cancers. Established CTC enumeration methods primarily rely on affinity capture of cell surface antigens, and have been criticized for underestimation of CTC numbers due to antigenic bias. Emerging CTC capture strategies typically distinguish these cells based on their assumed biomechanical characteristics, which are often validated using cultured cancer cells. In this study, we developed a software tool to investigate the morphological properties of CTCs from patients with castrate resistant prostate cancer and cultured prostate cancer cells in order to establish whether the latter is an appropriate model for the former. We isolated both CTCs and cultured cancer cells from whole blood using the CellSearch® system and examined various cytomorphological characteristics. In contrast with cultured cancer cells, CTCs enriched by CellSearch® system were found to have significantly smaller size, larger nuclear-cytoplasmic ratio, and more elongated shape. These CTCs were also found to exhibit significantly more variability than cultured cancer cells in nuclear-cytoplasmic ratio and shape profile.     

Ultra-rare-event detection performance of a custom scanning cytometer on a model preparation of fetal nRBCs

BACKGROUND: The performance of a fully automated scanning cytometer incorporating previously reported high-precision autofocus and accurate image segmentation was evaluated for the detection of "ultra-rare" cells using a model of fetal nucleated red blood cells (fnRBCs) in the maternal circulation. These distinctive scanning cytometry techniques were expected to markedly improve sensitivity and specificity. METHODS: Normal adult blood and fetal red blood cells were stained with fluorescein isothiocyanate-conjugated anti-fetal hemoglobin and 4,6-diamidino-2-phenylindole, a nuclear dye. Adult cells were spiked with fetal cells to create ratios of about 1 fnRBC in 10(7) nucleated cells and deposited in monolayers on slides using centrifugal cytology. Rare-event performance parameters were reviewed, formalized, and applied to test the new instrument using this cell model. RESULTS: Fifteen slides were analyzed to establish performance by comparison with manual detection, and four sets of four slides each were then scanned to explore the limit of detection. Results were an average sensitivity of 91%, an average specificity error of 12.3 false-positives per million cells, and repeatability of 100% at a cell analysis rate of 862 Hz. With addition of a quick interactive step subsequent to scanning, the false-positive rate dropped to a total of only one artifact over the 15 experiments. The instrument succeeded at locating 1 fnRBC in 20 million adult cells, the lowest limit of detection tested. CONCLUSION: This consistently high performance, coupled with the capability of scanning arbitrarily large numbers of cells, validates the considerable potential of precise high-speed autofocus and accurate real-time image segmentation for ultra-rare event detection.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4′,6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

Synthesis and evaluation of cytotoxicity of 6-amino-4-aryl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitriles

Several derivatives of 6-amino-4-aryl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitriles were synthesized via Biginelli type reaction and tested for their anti-proliferative activity on human breast cancer (MCF-7) and human colon carcinoma (HT29) cell lines. Malignant and non-malignant cells were cultivated in RPMI medium and incubated with different concentrations of these pyrimidines. Cell viability was evaluated by MTT assay. Apoptotic cells were determined using DAPI (4'-6-diamidino-2-phenylindole) and propidium iodide staining of DNA fragmentation by flow cytometry (sub-G1 peak). 6-Amino-4-(4-chlorophenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile and 6-amino-4-[4-dimethylamino)phenyl]-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile decreased the viability of MCF-7 and HT29 cells, in contrast to L929 cells. These compounds induced a sub-G1 peak inflow cytometry histograms of treated cells indicating that apoptosis is involved in their toxicity.     

miR-182在前列腺癌组织中的表达及其对前列腺癌细胞增殖和凋亡的影响

目的:观察前列腺癌组织及不同前列腺癌细胞系中miR-182的表达,并探讨下调其表达对前列腺癌细胞增殖和凋亡的影响及机制。方法:采用实时荧光定量PCR(q RT-PCR)检测30例前列腺癌组织和30例相应的癌旁组织以及前列腺正常上皮RWPE-1细胞、前列腺癌PC-3、LNCa P和DU145细胞中miR-182的表达,进一步采用Lipfectamine 2000脂质体转染miRNA-182 inhibitor和阴性对照miRNA于PC-3细胞后,通过噻唑蓝(MTT)比色法检测细胞增殖情况,流式细胞术检测细胞凋亡率,免疫印迹(Western blot)法检测转录因子FOXO1、血管内皮生长因子(VEGF)和抑癌基因p53蛋白的表达。结果:miR-182在前列腺癌组织中的表达明显高于癌旁组织(P0.05);miR-182在前列腺癌细胞系PC-3、LNCa P和DU145中的表达均高于前列腺正常上皮细胞RWPE-1(P0.05),其中PC-3细胞中miR-182表达水平最高。转染miRNA-182 inhibitor至PC-3细胞成功下调miR-182表达后,细胞的增殖能力明显受到抑制,细胞凋亡能力明显增强,FOXO1表达水平显著升高,VEGF和p53的表达明显降低,差异均具有统计学意义(P0.05)。结论:miR-182在前列腺癌组织及细胞中呈高表达,下调miR-182的表达可能通过增加FOXO1的表达并减少VEGF和p53的表达,抑制前列腺癌细胞增殖并诱导细胞凋亡。