Laser Scanning Cytometer Research Articles

Rapid and accurate evaluation of vaporized hydrogen peroxide on the efficiency of disinfection, using a sensitive dual-channel laser scanning cytometer

Vaporized hydrogen peroxide (VHP) is a highly active disinfectant, and VHP decontamination systems have been widely applied in hospitals, microbiological laboratories, and pharmaceutical industries. However, the decomposition of VHP into non-toxic by-products is essential. Evaluation of the disinfection efficacy of VHP is crucial to ensuring the reliability of VHP disinfection and controlling microbial contamination. In this study, a rapid and sensitive strategy is proposed to evaluate the efficacy of VHP in surface disinfection by detecting the survived and killed bacteria from VHP-exposed biological indicators (BIs). A dual-channel solid-phase cytometer is designed, and fluorescent dyes are used as indicators to automatically and accurately distinguish live cells from dead cells in the mixtures of bacteria. To verify the availability and effectiveness of the laser scanning cytometry, experiments on its application in estimating the efficacy of VHP disinfection practice have been carried out in this study, and its estimation effect compared with that of the traditional plate counting method. Results show that the proposed assay might distinctly identify live or killed cells labeled by green and red fluorescent dyes and examined the disinfection efficacy in 30 min by calculating the bactericidal rate. Compared with the plate counting method, the proposed approach is accurate and practical, with an average detection efficiency of 98.47% ± 1.55%. Moreover, an excellent correlation between the concentrations of B. subtilis var niger (ATCC 9372) measured by the proposed detection system and by the plate counting method is noticed (R2 = 0.9971), indicating that this approach had advantages in the detection of trace microorganisms. To summarize, the proposed strategy appears practical and significant in many fields in which microbial counting and identification are required.

Mitochondrial Network and Biogenesis in Response to Short and Long-Term Exposure of Human BEAS-2B Cells to Aerosol Extracts from the Tobacco Heating System 2.2.

Adverse effects of cigarette smoke on health are widely known. Heating rather than combusting tobacco is one of strategies to reduce the formation of toxicants. The sensitive nature of mitochondrial dynamics makes the mitochondria an early indicator of cellular stress. For this reason, we studied the morphology and dynamics of the mitochondrial network in human bronchial epithelial cells (BEAS-2B) exposed to total particulate matter (TPM) generated from 3R4F reference cigarette smoke and from aerosol from a new candidate modified risk tobacco product, the Tobacco Heating System (THS 2.2). Cells were subjected to short (1 week) and chronic (12 weeks) exposure to a low (7.5 µg/mL) concentration of 3R4F TPM and low (7.5 µg/mL), medium (37.5 µg/mL), and high (150 µg/mL) concentrations of TPM from THS 2.2. Confocal microscopy was applied to assess cellular and mitochondrial morphology. Cytosolic Ca2+ levels, mitochondrial membrane potential and mitochondrial mass were measured with appropriate fluorescent probes on laser scanning cytometer. The levels of proteins regulating mitochondrial dynamics and biogenesis were determined by Western blot. In BEAS-2B cells exposed for one week to the low concentration of 3R4F TPM and the high concentration of THS 2.2 TPM we observed clear changes in cell morphology, mitochondrial network fragmentation, altered levels of mitochondrial fusion and fission proteins and decreased biogenesis markers. Also cellular proliferation was slowed down. Upon chronic exposure (12 weeks) many parameters were affected in the opposite way comparing to short exposure. We observed strong increase of NRF2 protein level, reorganization of mitochondrial network and activation of the mitochondrial biogenesis process. Comparison of the effects of TPMs from 3R4F and from THS 2.2 revealed, that similar extent of alterations in mitochondrial dynamics and biogenesis is observed at 7.5 µg/mL of 3R4F TPM and 150 µg/mL of THS 2.2 TPM. 7 days exposure to the investigated components of cigarette smoke evoke mitochondrial stress, while upon chronic, 12 weeks exposure the hallmarks of cellular adaptation to the stressor were visible. The results also suggest that mitochondrial stress signaling is involved in the process of cellular adaptation under conditions of chronic stress caused by 3R4F and high concentration of THS 2.2.

Abstract P1-13-02: Early change in topoisomerase 1 (Top1) positive circulating tumor cells (CTCs) is associated with overall survival (OS) in patients with advanced breast cancer after treatment with etirinotecan pegol

Abstract Background: Etirinotecan pegol (EP) is a long-acting Top1 inhibitor providing sustained levels of active metabolite throughout the entire chemotherapy cycle. The phase 3 BEACON trial compared EP to treatment of physician's choice (TPC) in patients with advanced breast cancer, demonstrating a non-statistically significant 2.1 month difference in survival favoring EP in the intent to treat population. A novel aspect of the BEACON trial is to explore the utility of biomarkers measured in CTCs for predicting efficacy with EP. Pre- and post-treatment CTCs were isolated from blood of 77% of the 852 BEACON patients. Target-specific pharmacodynamic biomarkers for EP measured in CTCs were analyzed to identify patients most responsive to treatment with EP. Methods: Donation of blood samples for CTC analysis was voluntary. Participating BEACON patients had serial (baseline, Cycle 2 Day 1 [C2D1], Cycle 4 Day 1 [C4D1], End of Treatment) 7.5-mL whole blood samples drawn in EDTA tubes and shipped within 96 hours ambient to ApoCell (Houston, TX) for processing. PBMCs were separated by Ficoll® gradient, and CTCs were isolated using ApoStream® technology. Isolated cells were deposited on three slides and stained for DAPI, CD45, cytokeratin markers, as well as Top1, Top2, Ki67, γH2AX, Rad51, ABCG2, and TUNEL. Biomarkers were quantified by iCys® laser scanning cytometer equipped with image analysis software, and correlated with OS using Cox multiple regression and Kaplan-Meier analyses. Results: The CTC substudy yielded 611 pre-treatment, 519 C2D1, 268 C4D1, and 431 End of Treatment samples. Among the successfully processed blood samples, 98% had detectable CTCs, with a median of 63, 46, 51, and 57 CTCs/mL at baseline, C2D1, C4D1, and End of Treatment, respectively. Cox regression analyses of CTC number and percentage of Top1, Top2, Ki67, or TUNEL positive CTCs identified a correlation for post-treatment number of Top1-positive CTCs with OS in patients receiving EP. To assess the impact of Top1-positive CTCs, patients were classified as Top1-High (> median) or Top1-Low (≤ median) based on the percent of Top1-positive CTCs at baseline. Among the Top1-High patients at baseline, significantly improved OS (HR 0.54, p=0.007) was observed for those who converted to Top1-Low after their first treatment with EP (C2D1), but not TPC (HR 1.12, p=0.613). These results suggest that decreased number of Top1-positive CTCs may reflect EP target engagement with Top1, as these patients derived the most benefit from treatment. Conclusions: CTC collection and analysis was successfully incorporated into the phase 3 BEACON study, with 77% patient participation. CTC detection rate using ApoStream® was high, permitting evaluation of biomarkers at baseline and post-treatment. Significantly improved OS was observed in patients who had a decreased number of Top1-positive CTCs following cycle 1 of EP. Citation Format: Rugo HS, Cortes J, Awada A, O'Shaughnessy J, Twelves C, Im S-A, Hannah AL, Lu L, Sy S, Caygill K, Zajchowski D, Davis DW, Hoch U, Perez EA. Early change in topoisomerase 1 (Top1) positive circulating tumor cells (CTCs) is associated with overall survival (OS) in patients with advanced breast cancer after treatment with etirinotecan pegol. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-13-02.

Abstract P3-10-03: Etirinotecan pegol target-specific pharmacodynamic biomarkers in circulating tumor cells from patients with metastatic breast cancer in the phase 3 BEACON study

Abstract Background: Etirinotecan pegol (EP) is the first long-acting topoisomerase 1-inhibitor providing sustained level of active metabolite that concentrate in the tumor for an entire chemotherapy cycle. In a Phase 2 study (Lancet Oncology 2013), EP demonstrated a 29% overall response rate in patients with metastatic breast cancer, leading to the Phase 3 global BEACON study in this population. Detection of circulating tumor cells (CTCs) using immunomagnetic EpCAM-based methods have been conceptually accepted as a "liquid tumor biopsy". To circumvent the limited recovery of CTCs these methods provide for molecular profiling applications, we isolated CTCs based on an antibody-independent, continuous flow dielectrophoresis (DEP) technology (ApoStream®). We developed quantitative multiplex immunofluorescent assays for target-specific pharmacodynamics (PD) biomarkers for EP in CTCs isolated pre- and post-treatment. Here we present the distribution of the PD biomarkers in CTC samples collected from BEACON patients. Methods: Donation of blood samples for CTC analysis was voluntary. BEACON patients participating in the substudy had serial (pre-dose, Cycle 2 Day 1, Cycle 4 Day 1, End of Treatment) 7.5-mL whole blood samples drawn in EDTA tubes and shipped ambient to ApoCell (Houston, TX) within 96 hrs for processing. PBMCs were separated by Ficoll® gradient, and CTCs were isolated using ApoStream® technology. Isolated cells were deposited on three slides and stained for DAPI, CD45, and cytokeratin markers, as well as 2-3 of the seven PD markers, and analyzed by iCys® laser scanning cytometer equipped with image analysis software. CTC count, mean fluorescence intensity (MFI) of PD biomarkers, and % marker-positive cells were analyzed for their distribution. Results: 75% of the 852 BEACON patients participated in the CTC substudy, yielding 649 pre-treatment CTC samples. As of Mar14, 538 Cycle 2 Day 1, 281 Cycle 4 Day 1, and 394 End of Treatment samples were collected. 98% of pre-treatment blood samples were successfully processed. 97% had detectable CTCs, with a median of 504 CTCs (interquartile range: 128-1432). PD markers could be analyzed in 83-92% of samples after implementing a minimum requirement of 10 CTCs analyzed. Staining was positive for Top1, Top2, μH2Ax, Rad51, ABCG2, Ki67, and TUNEL in 82%, 71%, 14%, 30%, 20%, 56%, and 94% of samples, respectively. Percent of marker-positive cells varied from 0.2-69%, with 6- to 37-fold interquartile ranges. Median MFI in these cells varied between 25x103 and 100x103, with 1.2- to 1.6-fold interquartile ranges. Conclusions: CTC collection was successfully incorporated into the BEACON study, with 75% patient participation. CTC detection rate using ApoStream® was high, permitting evaluation of biomarkers at baseline and over time. EP target-specific PD biomarkers can be measured in CTCs isolated from patients participating in BEACON and will be assessed for their potential to predict clinical response. Final BEACON efficacy and safety results are expected in early 2015, which will allow further analysis of CTCs with patient outcome (response, PFS, OS) and change in CTCs at time of progression. Citation Format: Edith A Perez, Katie Caygill, Alison L Hannah, Javier Cortes, Ahmad Awada, Joyce O'Shaughnessy, Christopher Twelves, Hope Rugo, Seock-Ah Im, Kenna Anderes, Darren W Davis, Ute Hoch. Etirinotecan pegol target-specific pharmacodynamic biomarkers in circulating tumor cells from patients with metastatic breast cancer in the phase 3 BEACON study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-10-03.

Abstract P4-01-10: Predictive impact of circulating tumor cells with an epithelial-to-mesenchymal transition phenotype in patients with primary breast cancer treated with primary systemic therapy

Abstract Background: Tumor cells with a mesenchymal phenotype include cancer stem-like cells (CSCs), which are known to contribute to metastasis. Circulating tumor cells (CTCs) are epithelial cells in peripheral blood that are detected using an anti-EpCAM antibody; however, CTCs undergoing epithelial-mesenchymal transition (EMT) may not be detected using this method. We have developed an antibody-independent CTC enrichment platform, Apostream®, which does not rely on EpCAM-based capture. We used this instrument to determine the clinical relevancy and feasibility of measuring EMT-CTCs in breast cancer patients. Methods: Blood samples from newly diagnosed breast cancer patients were prospectively collected at baseline (T0), after primary systemic therapy (T1), and after definitive surgery (T2) and processed using the Apostream® system. Isolated cells were stained with antibodies to cytokeratin (anti-CK), leukocytes (anti-CD45), and the nuclear stain, 4,6-diamidino-2-phenylindole (DAPI), to identify CTCs. These CTCs were also stained with additional markers and examined on a laser scanning cytometer to measure protein expression levels of epithelial (EpCAM, E-cadherin), mesenchymal (β-catenin, vimentin) and CSC-markers (CD44, CD24). Pathological complete response (pCR) and residual cancer burden (RCB) statuses after preoperative treatment were obtained to correlate baseline CTCs and marker expression with treatment response. Results: The study enrolled 33 patients (10 with early-stage, 19 with locally advanced, and 4 with metastatic breast cancer); 32, 12, and 10 patients provided samples at T0, T1, and T2, respectively. Of the 20 patients who underwent surgery, 6 patients achieved pCR. CTCs were detected (≥ 1 cell in at least one of the three samples) in 47%, 75%, and 80% of the T0, T1, and T2 samples, respectively. EMT markers (either vimentin or β-catenin) were detected in 67%, 11%, and 38% of these CTCs, respectively. The mean number of CTCs per mL detected at T0 was 0.43 (range, 0-3.7). CTC detection was correlated with a high histological Nottingham index (P=0.039). Conversely, vimentin-positive CTC detection was inversely correlated with clinical stage at T0 (P=0.034). No significant correlation was observed between CTCs detected at baseline and breast cancer subtypes. However, there was a trend for CTCs detected at T0 to be predictive of chemotherapy response, as 62% of patients with CTCs at T0 achieved a pCR, whereas only 11% of patients without CTCs at T0 had a pCR (P=0.057). The other EMT and CSC markers we tested did not predict response. Conclusions: Apostream® was successful in detecting EMT-CTCs in this prospective study. There was a trend for the presence of CTCs at baseline to predict pCR. We will present our final data analysis of 50 patients. pCR (n=6)†RCB-I (n=2)RCB-II (n=5)RCB-III (n=4)P-valueCK+CD45- CTCsNegative (n=9)1(11)2(22)3(33)3(33)0.146 0.057*Positive (n=8)5(62)0(0)2(25)1(13)† All data are no. of patients (%) Two did not have an RCB status available, and 1 did not have a T0 sample. * Comparing pCR status (pCR vs no-pCR) between CTCs negative and positive. Citation Format: Fanny Le Du, Dzifa Y Duose, Elisha J Dettman, Jackson A Summer, Mariana Chavez-MacGregor, Carlos H Barcenas, Abenaa M Brewster, Alvarez H Ricardo, Vincente Valero, Ana M Gonzalez-Angulo, James M Reuben, Naoto T Ueno. Predictive impact of circulating tumor cells with an epithelial-to-mesenchymal transition phenotype in patients with primary breast cancer treated with primary systemic therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-10.

A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting.

BackgroundRadiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening.MethodsWe developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells.ResultsOn 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated.ConclusionsWe developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid the identification of molecular targets for radiosensitization, thereby contributing to improving the efficacy of radiotherapy.

PARP-1 expression in CD34+ leukemic cells in childhood acute lymphoblastic leukemia: relation to response to initial therapy and other prognostic factors.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein that impacts DNA repair and apoptosis. Both experimental and ongoing clinical studies indicate that PARP-1 inhibitors are potent and promising anticancer agents. However, the outcome of treatment with PARP-1 inhibitors depends on the expression of PARP-1 protein in the tumor cells. This study aimed to assess PARP-1 expression in peripheral blood CD34+ leukemic cells before and after 12 hours of prednisone administration as well as the relation between PARP-1 expression and early treatment response to initial therapy and other prognostic factors (immunophenotype, age, initial peripheral blood white blood count [WBC], and risk factor group). The study comprised 43 children with de novo ALL. Cytospins of peripheral blood were stained with mouse anti-CD34-FITC and anti-PARP-1 antibody followed by goat anti-mouse APC-conjugated antibody. DNA was counterstained with PI (propidium iodide). Cellular fluorescence was measured by a laser scanning cytometer. Statistically significant differences in baseline PARP-1 expression with respect to early treatment response (good vs. poor), ALL immunophenotype (ALL B vs. ALL T), age (children < 1 years and > 6 years vs. children 1-6 years), initial WBC (< 20 000/µl vs. ≥ 20 000/µl), and risk factor group (SR vs. IR vs. HR) were not found. PARP-1 expression was increased 12 hours after treatment in poor early treatment responders, whereas it remained statistically unchanged with respect to ALL immunophenotype, age, initial WBC, risk factor group and early treatment response. The overexpression of PARP-1 in poor early treatment responders suggests that it may contribute to treatment failure in this group of children with ALL. Our observation - if confirmed by other studies - may form the rationale for administration of PARP inhibitors in selected subsets of ALL children.

Agonist-induced β2-adrenoceptor desensitization and downregulation enhance pro-inflammatory cytokine release in human bronchial epithelial cells

It is not clear whether increased asthma severity associated with long-term use of β2-adrenoceptor (β2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between β-AR agonist-mediated effects on β2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o− cells grown in the presence and absence of β-AR agonists and/or antagonists, the β2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in β2-AR density. Both Salbu and ISO reduced cAMP generation in a concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the β2-AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered β2-AR expression and function in airway epithelial cells. β2-AR desensitization and downregulation induced by long-term treatment with β2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy.

Morphometry to identify subtypes of leukocytes

Recent studies in image cytometry evaluated the replacement of specific markers by morphological parameters. The aim of this study was to develop and evaluate a method to identify subtypes of leukocytes using morphometric data of the nuclei. The analyzed images were generated with a laser scanning cytometer. Two free programs were used for image analysis and statistical evaluation: Cellprofiler and Tanagra respectively. A sample of leukocytes with 200 sets of images (DAPI, CD45 and CD14) was analyzed. Using feature selection, the 20 best parameters were chosen to conduct cross-validation. The morphometric data identified the subpopulations of the analyzed leukocytes with a sensitivity and specificity of 0.95 per sample. The present study is the first that identifies subpopulations of leukocytes by nuclear morphology.

An HTS-Compatible 3D Colony Formation Assay to Identify Tumor-Specific Chemotherapeutics

There has been increasing interest in the development of cellular behavior models that take advantage of three-dimensional (3D) cell culture. To enable assessment of differential perturbagen impacts on cell growth in 2D and 3D, we have miniaturized and adapted for high-throughput screening (HTS) the soft agar colony formation assay, employing a laser-scanning cytometer to image and quantify multiple cell types simultaneously. The assay is HTS compatible, providing high-quality, image-based, replicable data for multiple, co-cultured cell types. As proof of concept, we subjected colorectal carcinoma colonies in 3D soft agar to a mini screen of 1528 natural product compounds. Hit compounds from the primary screen were rescreened in an HTS 3D co-culture matrix containing colon stromal cells and cancer cells. By combining tumor cells and normal, nontransformed colon epithelial cells in one primary screening assay, we were able to obtain differential IC50 data, thereby distinguishing tumor-specific compounds from general cytotoxic compounds. Moreover, we were able to identify compounds that antagonized tumor colony formation in 3D only, highlighting the importance of this assay in identifying agents that interfere with 3D tumor structural growth. This screening platform provides a fast, simple, and robust method for identification of tumor-specific agents in a biologically relevant microenvironment.

Etirinotecan pegol (EP) target-specific pharmacodynamic (PD) biomarkers measured in circulating tumor cells (CTCs) from patients in the phase III BEACON study in patients with metastatic breast cancer (mBC).

1087 Background: EP is a unique topoisomerase 1 inhibitor that provides continuous exposure to SN38. EP demonstrated a 29% overall response rate in patients with mBC, leading to a phase III global BEACON study in patients with mBC. CTCs in patient blood samples provide a minimally invasive approach to detect PD markers of drug activity. We developed quantitative multiplex immunofluorescent assays to measure target-specific PD biomarkers for EP in CTCs isolated pre- and post-treatment. Methods: Assays for Top1, Top2, g-H2Ax, Rad51, ABCG2, and Ki-67 were developed using control (0.1% DMSO) and drug-treated (SN38, 10 uM) tumor cell lines (HCT116, MCF7, A549, SKBr3) and PBMCs from healthy donors. The optimal antibody for each biomarker was then multiplexed in a panel with antibodies against cytokeratin, CD45 and DAPI for phenotypic identification of CTCs. For analysis of BEACON pts, serial 7.5 mL whole blood samples were drawn and shipped ambient to Apocell (Houston, TX) for further processing. PBMCs were separated and CTCs were isolated using ApoStream technology. CTCs were stained for PD markers and analyzed using an iCys laser scanning cytometer equipped with image analysis software. Results: Antibodies bound to tumor cells showed staining confined to the nucleus (Top1, Top2, g-H2AX, Ki-67) or membrane (ABCG2), exhibited defined peak separation from their isotype controls, and signal strength correlated with cellular expression of high and low levels of markers. To date, ~ 80% of BEACON pts consent to participate in the CTC sub-study. As of 30 Oct 2012, data from 167 pre-dose blood samples from BEACON pts were available. 99% of blood samples were successfully processed. 93% had detectable CTCs, yielding a median of 216 CTCs (range: 7.5-14816). Staining was positive for Top1, Top2, g-H2Ax, Rad51, ABCG2, and Ki-67 in 82%, 89%, 16%, 53%, 31%, 52% of samples, respectively. Conclusions: EP target-specific pharmacodynamic biomarkers can be reliably measured in CTCs isolated from patients participating in BEACON. Sample collection and analysis of pre- and post-treatment samples is ongoing. Clinical trial information: NCT01492101.

The effects of particle size on the deposition of fluorescent nanoparticles in porous media: Direct observation using laser scanning cytometry

The effects of particle size on the deposition of fluorescent nanoparticles in porous media were investigated using a microfluidic flow cell in conjunction with a laser scanning cytometer (LSC). All nanoparticle sizes investigated, i.e., 57 nm, 210 nm, and 510 nm particles, were found to attach to both the upstream and downstream surfaces of the collector, indicating that diffusion is a controlling mechanism for the transport of nanoparticles in this size range. Under an unfavorable condition (3mM NaCl, pH 7), the 510nm particles achieved a maximum retention capacity after the injection of 40PV, 0.0025% particle suspensions; however, the retention capacity was not yet achieved for the 57nm particles after the injection of 300PV, 0.0025% particle suspensions. Under a favorable condition (100mM NaCl, pH 7), the 57nm particles seemed to be more sensitive to changes in the water chemistry, showing more obvious mobilization and reattachment after the injection of DI water. The size dependent attachment rate and size dependent sensitivity to the surface heterogeneity are considered to contribute to the observed different behaviors of these particles.

Pore-Scale Investigation of Nanoparticle Transport in Saturated Porous Media Using Laser Scanning Cytometry

Knowledge of nanoparticle transport and retention mechanisms is essential for both the risk assessment and environmental application of engineered nanomaterials. Laser scanning cytometry, an emerging technology, was used for the first time to investigate the transport of fluorescent nanoparticles in a microfluidic flow cell packed with glass beads. The laser scanning cytometer (LSC) was able to provide the spatial distribution of 64 nm fluorescent nanoparticles attached in a domain of 12 mm long and 5 mm wide. After 40 pV of injection at a lower ionic strength condition (3 mM NaCl, pH 7.0), fewer fluorescent nanoparticles were attached to the center of the flow cell, where the pore-scale velocity is relatively higher. After a longer injection period (300 PV), more were attached to the center of the flow cell, and particles were attached to both the upstream and downstream sides of a glass bead. Nanoparticles attached under a higher ionic strength condition (100 mM NaCl, pH 7.0) were found to be mobilized when flushed with DI water. The mobilized particles were later reattached to some favorable sites. The attachment efficiency factor was found to reduce with an increase in flow velocity. However, torque analysis based on the secondary energy minimum could not explain the observed hydrodynamic effect on the attachment efficiency factor.

Degradation of p21Cip1 through Anaphase-promoting Complex/Cyclosome and Its Activator Cdc20 (APC/CCdc20) Ubiquitin Ligase Complex-mediated Ubiquitylation Is Inhibited by Cyclin-dependent Kinase 2 in Cardiomyocytes

Cyclin-dependent kinase inhibitor p21Cip1 plays a crucial role in regulating cell cycle arrest and differentiation. It is known that p21Cip1 increases during terminal differentiation of cardiomyocytes, but its expression control and biological roles are not fully understood. Here, we show that the p21Cip1 protein is stabilized in cardiomyocytes after mitogenic stimulation, due to its increased CDK2 binding and inhibition of ubiquitylation. The APC/CCdc20 complex is shown to be an E3 ligase mediating ubiquitylation of p21Cip1 at the N terminus. CDK2, but not CDC2, suppressed the interaction of p21Cip1 with Cdc20, thereby leading to inhibition of anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20)-mediated p21Cip1 ubiquitylation. It was further demonstrated that p21Cip1 accumulation caused G2 arrest of cardiomyocytes that were forced to re-enter the cell cycle. Taken together, these data show that the stability of the p21Cip1 protein is actively regulated in terminally differentiated cardiomyocytes and plays a role in inhibiting their uncontrolled cell cycle progression. Our study provides a novel insight on the control of p21Cip1 by ubiquitin-mediated degradation and its implication in cell cycle arrest in terminal differentiation.

A rapid detection method for apoptosis and necrosis measurement using the Cellometer imaging cytometry

Apoptosis and necrosis play an important role in various aspects of preclinical pharmaceutical drug discovery and validation. The ability to quickly determine the cytotoxic effect of chemical compounds on cancer cells allows researchers to efficiently identify potential drug candidates for further development in the pharmaceutical discovery pipeline. Recently, a new imaging cytometry system has been developed by Nexcelom Bioscience LLC (Lawrence, MA, USA) for fluorescence-based cell population analysis. Currently, fluorescence-based cell death assays have not been demonstrated by the Cellometer system, which can potentially provide a quick, simple, and inexpensive alternative method for smaller biomedical research laboratories. In this study, we demonstrate for the first time the use of Cellometer imaging cytometry for necrosis/apoptosis detection by studying the dose-response effect of heat and drug-induced cell death in Jurkat cells labeled with annexin V-FITC (apoptotic) and propidium iodide (necrotic). The experimental results were evaluated to validate the imaging cytometric capabilities of the Cellometer system as compared to the conventional flow cytometry. Similar cell population results were obtained from the two methods. The ability of Cellometer to rapidly and cost-effectively perform fluorescent cell-based assays has the potential of improving research efficiency, especially where a flow or laser scanning cytometer is not available or in situations where a rapid analysis of data is desired.

The use of HaloTag-based technology in flow and laser scanning cytometry analysis of live and fixed cells

BackgroundCombining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells.FindingsHere, we describe development of applications using protein tag technology (HaloTag (HT)-based) for flow and laser scanning cytometry (LSC). Cell lines, expressing recombinant surface β1-integrin-HT and HT-p65 fusion protein, and a CD4 T cell line (Jurkat) infected with human immunodeficiency virus type 1 (HIV-1) reporter virus expressing the unfused HT (HIV-1Lai-Halo), were stained with different HT ligands and successfully detected by flow cytometers equipped with 488 and 561 nm lasers as well as a laser scanning cytometer (equipped with 488 and 405 nm lasers) alone or combined with cell cycle and viability markers.ConclusionsUse of HT technology for cytometric applications has advantages over its use in microscopy as it allows for the statistical measurement of protein expression levels in individual cells within a heterogeneous cell population in combination with cell cycle analysis. Another advantage is the ability of the HaloTag to withstand long fixation and high concentration of fixative, which can be useful in research of infectious agents like HIV and/or mycobacteria.

The potential of laser scanning cytometry for early warning of algal blooms in desalination plant feedwater

When algal blooms (such as red tide) occur, early detection of increased algal growth in intake water offers desalination plant operators advanced warning of severe fouling and provides the necessary time for preventative measures. Monitoring basic water quality measurements such as turbidity, silt density index (SDI), dissolved oxygen, and bulk fluorescence can approximate algal activity; however, these measurements lack the resolution needed to accurately track growth throughout all phases of a bloom event. This study investigates the use of a laser scanning cytometer (LSC) that detects algal cells over a wide range of concentrations (50–150,000 cells/ml), and characterizes cell shape, size, and distribution parameters through fluorescence signal imaging. Water samples are filtered to collect algae on a 0.2-μm membrane surface. Sample volume is variable so cells can be concentrated. The membrane surface is scanned with a 635-nm diode laser and the signal is processed to generate a cell count and distribution image. LSC performance was comparable to fluorescence microscopy and flow cytometry over a range of concentrations that may be encountered during a bloom event. The applicability of LSC was demonstrated using laboratory-grown cultures and seawater samples taken during a bloom event off of the coast of Long Beach, CA.

Abstract 3461: A chemical screen for the identification of novel therapeutics for pediatric brain tumors

Abstract Treatment of brain tumors in children has greatly improved over the past decades, but current treatment protocols lack specificity, killing not only tumor cells, but also “healthy” normal brain cells. Moreover, current therapy still fails in approximately 20% of medulloblastoma patients, 50% of ependymomas and 80% of children suffering from high grade glioma. Treatment mostly consists of surgery, followed by chemotherapy and radiotherapy, and although this kind of therapy is among the most effective treatment options available, it is not sufficient. Therefore, new therapeutic drugs are required that specifically kill brain tumor cells and/or sensitize to irradiation, sparing normal brain tissue. In order to identify such novel compounds, drug screens were set up in a semi-high throughput format, using Gaussia luciferase (Gluc) activity as cell viability readout. As opposed to other forms of luciferase, Gluc is secreted in the medium, allowing the measurement of luciferase activity without lysing the cells. Since the Gluc signal has been shown to be linearly related to cell number, this assay can be used to determine cell viability at different points after drug treatment. In addition, an Acumen eX3 laser scanning cytometer was used in parallel, to determine cell number and cytometric parameters. We screened two small molecule libraries (2960 compounds) for cell growth inhibitory or radiosensitizing properties on pediatric high-grade glioma, ependymoma and medulloblastoma cell lines and primary patient material. Cells were incubated with a concentration of 1µM of the different compounds, either as single agent or in combination with 3 Gray irradiation, and cell viability was determined over a period of 4 days of incubation. A reduction of &amp;gt;75% of cell growth was considered to be significant. In general, 4 different groups of small molecules could be distinguished: 1) compounds that did not affect cell proliferation compared to controls, 2) compounds that block cell cycle progression, 3) compounds that induce cell death independently of irradiation, and 4) radiosensitizers. A total of 114 compounds was identified that inhibited cell growth and/or sensitized towards irradiation. Among the compounds that induced cell death were microtubule inhibitors, alkylating agents, topoisomerase inhibitors and anthracyclins. Besides these known anti-cancer agents we also identified a large number of cardiotonic drugs, benzimidazoles and limonoids that effectively killed brain tumor cells. We are currently investigating these hits and expanding our drug screens with additional primary pediatric brain tumor cells. In addition, we are repeating the screens on non neoplastic primary human cells, including astrocytes, to assess the therapeutic index of these drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3461. doi:10.1158/1538-7445.AM2011-3461

Subcellular localization of p27 and prostate cancer recurrence: automated digital microscopy analysis of tissue microarrays

Previous investigations have linked decreased nuclear expression of the cell cycle inhibitor p27 with poor outcome in prostate cancer. However, these reports are inconsistent regarding the magnitude of that association and its independence from other predictors. Moreover, cytoplasmic translocation of p27 has been proposed as a negative prognostic sign. Given the cost and accuracy limitations of manual scoring, particularly of tissue microarrays, we determined if laser-based fluorescence microscopy could provide automated analysis of p27 in both nuclear and cytoplasmic locations and, thus, clarify its significance as a prognostic biomarker. We constructed tissue microarrays covering 202 recurrent cases (rising prostate-specific antigen) and 202 matched controls without recurrence. Quadruplicate tumor samples encompassed 5 slides and 1616 cancer histospots. Cases and controls matched on age, Gleason grade, stage, and hospital. We immunolabeled epithelial cytoplasm with Alexafluor 647, p27 with Alexafluor 488, and nuclei with 4c6-diamidino-2-phenylindole·2HCl. Slides were scanned on an iCys laser scanning cytometer (CompuCyte Corp, Cambridge, MA). Nuclear crowding required a stereological approach--random arrays of circles (phantoms) were layered on images and the content of each phantom was analyzed in scatter plots. Both nuclear and cytoplasmic p27 were significantly lower in cases versus controls (P = .014 and P = .004, respectively). Regression models controlling for matching variables plus prostate-specific antigen showed strong linear trends for increased risk of recurrence with lower p27 in both nucleus and cytoplasm (highest versus lowest quartile; odds ratio, 0.35; P = .006). Manual scoring identified an inverse association between p27 expression and tumor grade but no independent association with recurrence. In conclusion, we developed an automated method for subcellular scoring of p27 without the need to segment individual cells. Our method identified a strong relationship, independent of tumor grade, stage, and prostate-specific antigen, between p27 expression--regardless of subcellular location--and prostate cancer recurrence. This relationship was not observed with manual scoring.

Steps in metastasis research: Analyzing, collecting, and culturing circulating tumor cells

tumor cells (CTCs) have been in the focus ofcancer research for decades. The presence of these cells in theblood of patients with primary and metastatic cancer is asso-ciated with poor prognosis (1). CTCs have a central role in theprocess of metastasis formation: To become a CTC, cancercells must shed from the primary tumor by loosing cell–cellcontacts with other cancer cells. Furthermore, cancer cellsmust have the ability to migrate through the extracellular ma-trix and to invade the surrounding tissue. After intravasationinto the bloodstream, cancer cells became CTCs. In the blood-stream, a large fraction of the CTCs die quickly because of kill-ing by (i) the immune system; (ii) hemodynamic forces; and/or (iii) apoptosis evoked by loss of cell attachment and cell–matrix connections. Under a strong evolution pressure, CTCsdo accumulate lots of properties enabling them to survive inthe bloodstream and to extravasate the blood vessels. SomeCTCs extravasate into distant organs and persist there as dor-mant cells or are capable of dividing and forming micrometas-tases and later metastases (2).In the past decade, several CTC detection and enumera-tion methods have been developed. Immunolabeled cancercells from the blood can be enriched by different approachesincluding immunomagnetic separation and physical methods(filtration and density gradient). After enrichment, the iso-lated cancer cells can be enumerated and analyzed using differ-ent technical approaches (e.g., flow cytometer, laser scanningcytometer, and fluorescence microscope). Reverse transcrip-tion (RT-PCR)-based methods are also widely used. These arehighly sensitive for the expression of candidate genes specificto cancer cells or to the normal tissues from which the tumorcells originate (3). Unfortunately, these valuable methods arebarely usable to describe differences between CTC subpopula-tions.In a heterogenic CTC population, several CTC subpopu-lations might exist with different properties. Because cancercells having the ability to form micrometastases and metasta-ses are likely emerge from CTCs, a high medical need exists todevelop new methods to characterize the genotype and pheno-type of CTCs. According to our opinion, investigation of CTCsubpopulations might open new ways in anticancer research.Methods capable of studying CTC subpopulations might bevaluable tools not only for basic cancer research but also forcancer diagnostic, evaluation of prognosis, or following up theefficacy of anticancer therapy.In this issue, Takao and Takeda (page 107) describe a sim-plified protocol to enumerate CTCs without membrane-per-meabilization using a novel cross contamination free flow cy-tometer apparatus. The device has included a separate micro-fluidic chip, so using the new method CTCs can not only beenumerated with a cytometric multicolor analysis but also thedevice is able to collect both dead and viable circulating cancercells. The ratio of dead and live CTCs might have an impor-tance in clinical practice, for example, an increase in the ratiomight suggest a successful therapeutic outcome. We agree withthe authors that monitoring dead/live CTC ratio in additionto CTC number after adjuvant or neoadjuvant therapy couldprovide clinically relevant information. Furthermore, mostmethods which enumerate CTCs are not able to collect the