Automated Cell Imaging Research Articles

B-014 Performance evaluation of the automated cell image analyzer DI-60 for leukocyte differential count

Abstract Background The Sysmex DI-60 system (Sysmex, kobe, Japan) is an automated cell image analyzer that captures cell images and analyzes them. The aim of this study was to evaluate the performance of DI-60 for leukocyte differential count in comparison with manual differential count or XN-20. Methods A total of 205 samples were analyzed. The agreement between DI-60 pre-classification and post-verification by medical technicians was determined. The correlation and ability to identify clinically important abnormal cells of DI-60 post-verification were evaluated. Results The overall agreement of DI-60 pre-classification was 84.7% (Table 1). The correlation between DI-60 post-verification and manual differential counts were exellent (r2 > 0.85) for neutrophil, lymphocyte, monocyte and eosinophil, but was very low for basophil (r2 = 0.3759). The correlation between DI-60 post-verification and leukocyte differential counts by XN-20 were exellent (r2 > 0.90) for neutrophil, lymphocyte and eosinophil. However, monocyte and basophil showed a relatively low correlation of r2 = 0.6343 and r2 = 0.3118, respectively but was very low for basophil (r2 0.3759). The ability of DI-60 post-verification to identify clinically important abnormal cells including blast, promyelocyte, myelocyte, metamyelocyte and NRBC demonstrated excellent efficiency (80.8 to 96.3%) except for NRBC (51.0%). Overall sensitivity was 63.7-88.2%, and specificity was 84.3-95.5% excluding NRBC (43.7%). Conclusions DI-60 showed excellent pre-classification and generally high correlation compared to manual leukocyte differential counts. Although additional verification with PBS review by experienced medical technician may still be required, the performance DI-60 makes it an efficient screening tool in clinical laboratories.

Comparing automated cell imaging with conventional methods of measuring cell proliferation and viability

The ability to assess cell proliferation and viability is essential for assessing new drug treatments, particularly in cancer drug discovery. This study describes a new method that uses a plate reader digital microscopy cell imaging and analysis system to assess cell proliferation and viability. This imaging system utilizes high throughput fluorescence microscopy with two fluorescent probes: cell membrane-impermeable SYTOX green and nuclear binding Hoechst-33342. Here we compare this technology to other known viability assays, namely: propidium iodide (PI)-based flow cytometry, and sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based plate reader assays. These methods were assessed based on their effectiveness in detecting the cell numbers of two adherent cell lines and one suspension cell line. Automated cell imaging was most accurate at measuring cell number in both adherent and suspension cell lines. The PI-based flow cytometry method was more difficult to use with adherent cells, while the SRB and MTT assays had difficulties when monitoring cells in suspension. Despite these challenges, it was possible to obtain similar results when quantifying the effect of cytotoxic compounds. This study demonstrates that the digital microscopy automated cell imaging system is an effective method for assessing cell proliferation and the cytotoxic effect of compounds on both adherent and suspension cell lines.

Improving the power of drug toxicity measurements by quantitative nuclei imaging

Imaging-based anticancer drug screens are becoming more prevalent due to development of automated fluorescent microscopes and imaging stations, as well as rapid advancements in image processing software. Automated cell imaging provides many benefits such as their ability to provide high-content data, modularity, dynamics recording and the fact that imaging is the most direct way to access cell viability and cell proliferation. However, currently most publicly available large-scale anticancer drugs screens, such as GDSC, CTRP and NCI-60, provide cell viability data measured by assays based on colorimetric or luminometric measurements of NADH or ATP levels. Although such datasets provide valuable data, it is unclear how well drug toxicity measurements can be integrated with imaging data. Here we explored the relations between drug toxicity data obtained by XTT assay, two quantitative nuclei imaging methods and trypan blue dye exclusion assay using a set of four cancer cell lines with different morphologies and 30 drugs with different mechanisms of action. We show that imaging-based approaches provide high accuracy and the differences between results obtained by different methods highly depend on drug mechanism of action. Selecting AUC metrics over IC50 or comparing data where significantly drugs reduced cell numbers noticeably improves consistency between methods. Using automated cell segmentation protocols we analyzed mitochondria activity in more than 11 thousand drug-treated cells and showed that XTT assay produces unreliable data for CDK4/6, Aurora A, VEGFR and PARP inhibitors due induced cell size growth and increase in individual mitochondria activity. We also explored several benefits of image-based analysis such as ability to monitor cell number dynamics, dissect changes in total and individual mitochondria activity from cell proliferation, and ability to identify chromatin remodeling drugs. Finally, we provide a web tool that allows comparing results obtained by different methods.

Elesclomol-Cu Induces Cuproptosis in Human Acute Myeloid Leukemia Cells

To investigate the effects of elesclomol-Cu (ES-Cu) on the proliferation and cuproptosis of human acute myeloid leukemia (AML) cells. The effects of ES-Cu on the proliferation of AML cells and the AML cells pre-treated with ammonium tetrathiomolybdate (TTM) were examined by CCK-8 assay. The Calcein/PI kit was used to detected the changes in activity and cytotoxicity of AML cells induced by ES-Cu. Flow cytometry and Cytation3 fully automated cell imaging multifunctional detection system were used to analyze DCFH-DA fluorescence intensity, so as to determine the level of reactive oxygen species (ROS). The GSH and GSSG detection kits were used to measure the intracellular GSH content. Western blot was used to detected the expression of cuproptosis-related proteins ATP7B, FDX1, DLAT and DPYD. ES-Cu inhibited the proliferation of Kasumi-1 and HL-60 cells in a concentration-dependent manner (r Kasumi-1=-0.99， r HL-60=-0.98). As the concentration of ES-Cu increased, the level of intracellular ROS also increased (P <0.01-0.001). TTM could significantly reverse the inhibitory effect of ES-Cu on cell proliferation and its promoting effect on ROS. With the increase of ES-Cu concentration, the content of GSH was decreased (r =-0.98), and Western blot showed that the protein expressions of ATP7B, FDX1, DLAT and DPYD were significantly reduced (P <0.05). ES-Cu can induce cuproptosis in AML cells, which provides a new idea for the treatment of AML.

Esculin alleviates LPS-induced acute lung injury via inhibiting neutrophil recruitment and migration.

Acute lung injury (ALI) poses a serious threat to human health globally, particularly with the Coronavirus 2019 (COVID-19) pandemic. Excessive recruitment and infiltration of neutrophils is the major etiopathogenesis of ALI. Esculin, also known as 6,7-dihydroxycoumarin, is a remarkable compound derived from traditional Chinese medicine Cortex fraxini. Accumulated evidence indicates that esculin has potent anti-inflammatory effects, but its pharmaceutical effect against ALI and potential mechanisms are still unclear. This study evaluated the protective effect of esculin against ALI by histopathological observation and biochemical analysis of lung tissues and bronchoalveolar lavage fluid (BALF) in lipopolysaccharide (LPS)-challenged ALI mice in vivo. The effects of esculin on N-formyl-met-leu-phe (fMLP)-induced neutrophil migration and chemotaxis were quantitatively assessed using a Transwell assay and an automated cell imaging system equipped with a Zigmond chamber, respectively. The drug affinity responsive target stability (DARTS) assay, in vitro protein binding assay and molecular docking were performed to identify the potential therapeutic target of esculin and the potential binding sites and pattern. Esculin significantly attenuated LPS-induced lung pathological injury, reduced the levels of pro-inflammatory cytokines in both BALF and lung, and suppressed the activation of NF-κB signaling. Esculin also significantly reduced the number of total cells and neutrophils as well as myeloperoxidase (MPO) activity in the BALF. Esculin impaired neutrophil migration and chemotaxis as evidenced by the reduced migration distance and velocity. Furthermore, esculin remarkably inhibited Vav1 phosphorylation, suppressed Rac1 activation and the PAK1/LIMK1/cofilin signaling axis. Mechanistically, esculin could interact with β2 integrin and then diminish its ligand affinity with intercellular adhesion molecule-1 (ICAM-1). Esculin inhibits β2 integrin-dependent neutrophil migration and chemotaxis, blocks the cytoskeletal remodeling process required for neutrophil recruitment, thereby contributing to its protective effect against ALI. This study demonstrates the new therapeutic potential of esculin as a novel lead compound.

Mixed-phenotype acute leukemia, T/megakaryoblastic: does it really exist?

Mixed-phenotype acute leukemias (MPAL) account for < 4% of all cases of acute leukemias. These are a heterogeneous group of leukemias grouped together by the WHO classification as "rare subtypes." The diagnosis and treatment of MPAL is extremely challenging particularly for low middle income countries. Of these, B/myeloid and T/myeloid combinations are relatively common subtypes. However, megakaryoblastic and erythroid lineages in combination with other lineages are still rare enough to not even be addressed in the WHO classification. To date, there have been only a few reports of mixed B or T cell and megakaryocytic or mixed B or T cell and erythroid leukemias. We report the clinical presentation, diagnostic profile, and disease course of MPAL cases with a biphenotypic pattern consistent with T/megakaryoblastic lineage which is not yet defined in WHO classification. These cases were phenotyped using 8-color flow cytometry (BD FACS CANTO-II) using an extensive panel of markers. Interphase fluorescence in situ hybridization (FISH) was done using dual color dual fusion probes for BCR::ABL1, RUNX1::RUNX1T1, and ETV6::RUNX1, while MLL and CBFB gene rearrangement was tested by break-apart probes. Karyotyping was performed using the conventional GTG-banding technique. Both FISH and karyotyping were analyzed by the automated cell imaging system Leica Biosystems, using Cytovision MB8. The cases presented here satisfy the criteria for both T-lineage assignment (cyCD3 intensity reaches that of normal T-lymphocytes) and acute megakaryoblastic leukemia (≥ 1 megakaryocytic marker in > 50% blasts) and thus represent the first documented examples of this unusual entity from Pakistan. It is crucial to report these cases to gather more data about clinical presentation, diagnostic profile, and disease course. Additionally, the reported cases highlight the limitations of existing classifications which do not address rare subtypes. More importantly, T/megakaryoblastic MPAL needs to be included in the WHO classification as a separate entity.

Sub-monthly prediction of harmful algal blooms based on automated cell imaging

Harmful algal blooms (HABs) are an increasing threat to global fisheries and human health. The mitigation of HABs requires management strategies to successfully forecast the abundance and distribution of harmful algal taxa. In this study, we attempt to characterize the dynamics of 2 phytoplankton genera (Pseudo-nitzschia spp. and Dinophysis spp.) in Narragansett Bay, Rhode Island, using empirical dynamic modeling. We utilize a high-resolution Imaging FlowCytobot dataset to generate a daily-resolution time series of phytoplankton images and then characterize the sub-monthly (1–30 days) timescales of univariate and multivariate prediction skill for each taxon. Our results suggest that univariate predictability is low overall, different for each taxon and does not significantly vary over sub-monthly timescales. For all univariate predictions, models can rely on the inherent autocorrelation within each time series. When we incorporated multivariate data based on quantifiable image features, we found that predictability increased for both taxa and that this increase was apparent on timescales >7 days. Pseudo-nitzschia spp. has distinctive predictive dynamics that occur on timescales of around 16 and 25 days. Similarly, Dinophysis spp. is most predictable on timescales of 25 days. The timescales of prediction for Pseudo-nitzschia spp. and Dinophysis spp. could be tied to environmental drivers such as tidal cycles, water temperature, wind speed, community biomass, salinity, and pH in Narragansett Bay. For most drivers, there were consistent effects between the environmental variables and the phytoplankton taxon. Our analysis displays the potential of utilizing data from automated cell imagers to forecast and monitor harmful algal blooms.

Kinetic measurements of human CD8+ T cell cytotoxic activity in a 384-well plate format.

The elimination of infected or cancerous cells by CD8+ cytotoxic T lymphocytes (CTL) is a crucial effector mechanism of the immune system. Upon antigen recognition, CTL stop migrating, establish a tight contact with target cells and deliver cytotoxic molecules such as perforin and granzymes that lead to target cell apoptosis. The ability of CTL to control a population of infected cells or a tumor depends on multiple parameters, such as the relative numbers of CTL and target cells, the intrinsic cytotoxic activity of CTL, the intrinsic resistance of target cells and the repertoire of immune checkpoints tuning cytotoxic activity at the CTL:target cell interface. In this context, in vitro assays to precisely measure CTL:target cell interactions and cytotoxic activity over time are required to monitor natural or therapeutic responses. We here present an image-based method that allows recording of positions and survival of CTL and target cells over time in a high-throughput format. The protocol relies on the staining of CTL and target cells with fluorescent dyes and the automated imaging of cells deposited in wells of a 384-well plate with an automated cell imaging device. We discuss potential applications offered by the kinetic assessment of CTL cytotoxic activity in a high-throughput format.

Evaluation of new solution for cardiac graft preservation on cellular model

Evaluation of new solution for cardiac graft preservation on cellular model. Organ transplantation faces a growing shortage of grafts. To deal with this, surgeons have to consider organs from so-called “extended criteria” donors (elderly donors, smokers, donors with co-morbidities…) with increased sensitivity to ischemia-reperfusion injury. To improve preservation of these grafts, two approaches are possible: static preservation or dynamic preservation. Our laboratory has developed and patented a new solution, called LYPS (LYon Preservative Solution), intended for these two situations. The objective of the present study is to demonstrate the superiority of the LYPS compare to the other solutions currently used in clinical practice. In order to simultaneously study the effect of different solutions on cell conservation at different temperatures, we used a “Hypothermal Bioreactor”, specially developed for this work. We determined the impact of 6 different preservative solutions (LYPS, Celsior, UW, custodiol, Plegisol and IGL1) on H9C2 cells. We performed a 20 hours cold (5 °C) or mild hypothermic (20 °C) static preservation and 2 h of warm so-called “reoxygenation” (37 °C). Cell viability was assessed by MTT assay; and PI and Dilc1 staining determined cell necrosis and mitochondrial membrane potential by flow cytometry. We used a new automated cell imaging system (ImageXpress Pico) and metabolic biosensors to evaluate cytosolic NAD+ levels with SoNar and mitochondrial ATP levels modifications with ATeam-mito. It appears that, at the two temperatures studied (5 and 20 °C), the LYPS solution provides better viability at 5 and 20 °C (viability increased by 5.75 and 11.57 times compare to Plegisol). Necrosis after 20 h of preservation reached 17.3% and 13.54% at 5 and 20 °C with LYPS that is statically lower compared to the others solutions used. We also observed a smaller decrease of DYm with the use of LYPS solution. The levels of NAD and ATP were monitored and seem to be less impacted by the 20 hours of preservation in the presence of LYPS compared to the other solutions. All of these data show that the LYPS solution seems superior to the other solutions tested for the preservation of H9C2 cardiac cells, whether at 5 or at 20 °C. The following step is now to evaluate LYPS on heart preservation on porcine preclinical model.

Abstract P5-12-08: The role of a novel phosphatase NUDT5 in triple-negative breast cancer

Abstract Background: 281,550 new cases of invasive breast cancer will be diagnosed in US this year. 15% of these patients will be diagnosed with triple-negative breast cancers (TNBCs), which is the most aggressive subtype of breast cancer. Non-specific chemotherapy remains the current standard of care for TNBCs, due to the lack of targeted therapy. We hypothesized that phosphatases are potential new drug targets for TNBCs and performed a whole phosphatase microarray analysis using TNBCs vs. estrogen receptor (ER)-positive breast cancers. NUDT5, the enzyme hydrolyzes 8-oxo-dGDP and ADP-ribose, is among the most highly expressed phosphatases in TNBCs. The goal of these studies is to investigate NUDT5 dependency and mechanism in TNBC development.Methods: In this study, we have obtained NUDT5 mRNA expression level from publicly available TCGA, METABRIC, and the CCLE datasets, and compared NUDT5 mRNA level in TNBC vs. ER-positive breast cancer. Next, we examined the association of overall survival with NUDT5 expression level dichotomized by median mRNA expression in breast cancer patients. We then engineered doxycycline-inducible NUDT5 knockdown and knockout systems in TNBC cell lines, and characterized cell growth features over 7 days with or without NUDT5 depletion. Growth inhibition effect is also confirmed by NUDT5 inhibitor. After 72 hours of doxycycline induction, cell growth was determined by cell count over 7 days. Using these TNBC cellular models, we also determined molecular phenotypes after NUDT5 depletion by immunofluorescent microscopy for 8-oxo-dGDP and γH2AX. ImageXpress Automated Cell Imaging System and CellReporterXpress were applied for image processing and data quantification. Results: NUDT5 RNA is highly overexpressed in TNBCs as compared to ER-positive breast cancers and normal breast tissue. Survival analyses indicated that increasing NUDT5 level is significantly correlated with worse clinical outcomes of breast cancer patients. NUDT5 protein is highly expressed in TNBC cell lines, compared to ER-positive breast cancer cell lines. NUDT5 knockdown or knockout both significantly inhibits the growth of TNBC cell lines. Loss of NUDT5 induced the oxidative DNA lesion 8-oxoG level in the nucleus, as well as the ratio of γH2AX-positive cells. The inhibitor of NUDT5 also suppressed TNBC cell growth in vitro. Conclusions: Our results showed that the level of NUDT5 negatively correlates with TNBC patient survival. NUDT5 is required for the TNBC cells growth and tolerance to endogenous DNA damage agents. Our findings have revealed the essential function of NUDT5, and will provide critical insights for the future development of NUDT5 inhibitors for the treatment of TNBC patients. This work was supported by Charles Cain Endowment (PB). Citation Format: Jing Qian, William Tahaney, Yanxia Ma, Abhijit Mazumdar, Powel Brown. The role of a novel phosphatase NUDT5 in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-08.

A novel specific Aβ42 oligomer antibody ameliorates the neurotoxicity of patient‐derived brain extracts

AbstractBackgroundIn Alzheimer’s disease (AD), the neurotoxicity of amyloid‐β 1‐42 (Aβ42) stems mostly from soluble oligomeric aggregates of this peptide. However, the study of these aggregates has been hampered by their unstable and elusive nature, thus questioning the very definition of them as distinct targets for drug development. Here, we assess the toxicity of physiologically‐derived Aβ using extracts from deceased patients and controls in primary neuron cultures. By using a monoclonal antibody with a unique binding profile, ALZ‐201, we demonstrate that all of the Aβ toxicity observed in human‐derived brain extracts stem from soluble Aβ42 aggregates.MethodBrain tissue samples from 10 individuals were confirmed as AD (n=7) and non‐AD (n=3) cases using IHC staining for Aβ and p‐Tau aggregates. Extracts were prepared as described in Hong et al. 2018 (doi:10.1007/s00401‐018‐1846‐7) and immunodepleted with either the antibody 4G8 (removes all Aβ), ALZ‐201 (100% specific for non‐fibrillar soluble Aβ42 aggregates) or an isotype control to ALZ‐201 (does not bind any Aβ). Fractions were biochemically characterized, and toxicity assays were performed in primary mouse neuronal cultures using an automated cell imaging platform.ResultThe total Aβ content was higher in AD cases, as expected. The toxicity of the patient extracts exceeded those obtained from non‐AD cases with respect to neuronal loss and morphological determinants such as synapse density and measures of neurite complexity. Immunodepletion using 4G8 dramatically reduced Aβ in the AD patient samples and the control samples. This is in contrast to immunodepletion using ALZ‐201 or the isotype control, for which there was no detectable difference. Despite this remarkable difference in Aβ content, the therapeutic effect of depletion with 4G8 and ALZ‐201 was comparable, whereas there was no effect after depletion using the isotype control.ConclusionThe results demonstrate that very small amounts of soluble aggregated Aβ42 likely account for a large part of the toxicity in AD patients. The unique antibody, ALZ‐201, is capable of depleting these without targeting other forms of Aβ. Since this natural toxic form of Aβ is extremely low in abundance, this feature may be a critical attribute for achieving a true therapeutic effect in actual patients.

Specific targeting of a highly toxic subpopulation of Aβ42 oligomers for the treatment of Alzheimer’s disease

AbstractBackgroundNon‐clinical data on the etiology of Alzheimer’s disease (AD) suggest that only a very small fraction of soluble oligomeric Aβ42 carries most of the neurotoxic potency. But the study of Aβ42 soluble species has been hampered by their intrinsic heterogeneous and dynamic nature. A unique oligomer‐stabilizing technology, AβCC, enables the development of therapies that target these specifically. We here demonstrate the development such immunotherapies that exhibit unique Aβ oligomer targeting activity which translates into high efficacy in non‐clinical models of the disease.MethodStable synthetic Aβ42CC peptide oligomer derivatives were used as antigens in an active vaccine and were used to develop a murine monoclonal antibody, ALZ‐201. Efficacy was evaluated on brain extracts from AD patients and controls. These brain extracts were immunodepleted with ALZ‐201 and control antibodies. Toxicity assays were then performed in primary mouse neuronal cultures using an automated cell imaging platform. In addition, acute neurotoxicity effects on memory formation in zebrafish were assessed following intracranial injections of the brain extracts.ResultThe AβCC oligomers were able to generate truly oligomer‐specific antibodies. AD extracts exhibited higher levels of toxicity in contrast to non‐AD extracts in both cells assays and in zebrafish. This toxicity was highly dependent on Aβ, shown by immunodepletion with 4G8. Comparable toxicity was observed for brain extracts immunodepleted with ALZ‐201 as for extracts immunodepleted with 4G8. Interestingly, the soluble Aβ42 species captured by ALZ‐201 were below the limit of detection with the standard biochemical techniques used for analysis.ConclusionThe data support the hypothesis that a specific subtype of Aβ42 oligomers has large neurotoxic effects in the central nervous system that can affect synaptic plasticity, learning and memory. These oligomers are present in very small amounts and, due to the inaccessible environment, are not likely to be targeted with non‐specific anti‐Aβ immunotherapies. In contrast, targeting them specifically may serve as effective future treatments.

Evaluating the Performance of the Sysmex DI-60 Automated Cell Image Analyzer for the Differential Analysis of Leukocytes

Evaluating the Performance of the Sysmex DI-60 Automated Cell Image Analyzer for the Differential Analysis of Leukocytes

3D Microscopy Keeps Getting Faster, Smarter, Leaner

3D Microscopy Keeps Getting Faster, Smarter, Leaner

Unified Approach for White Blood Cell Segmentation, Feature Extraction, and Counting using Max-Tree Data Structure

Accurate identification and counting of White Blood Cells (WBCs) from microscopy blood cell images are vital for several blood-related disease diagnoses such as leukemia. The inevitability of automated cell image analysis in medical diagnosis results in a plethora of research for the last few decades. Microscopic blood cell image analysis involves three major steps: cell segmentation, classification, and counting. Several techniques have been employed separately to solve these three problems. In this paper, a simple unified model is proposed for White Blood Cell segmentation, feature extraction for classification, and counting with connected mathematical morphological operators implemented using the max-tree data structure. Max-tree creates a hierarchical representation of connected components of all possible gray levels present in an image in such a way that the root holds the connected components comprise of pixels with the lowest intensity value and the connected components comprise of pixels with the highest intensity value are in the leaves. Any associated attributes such as the size or shape of each connected component can be efficiently calculated on the fly and stored in this data structure. Utilizing this knowledge-rich data structure, we obtain a better segmentation of the cells that preserves the morphology of the cells and consequently obtain better accuracy in cell counting.

Multi-parametric Cell-Based Inflammation Assays for Cytokines and Cell Surface Antigens

Abstract Macrophages and vascular cells upregulate expression of cytokines and adhesion molecules in response to inflammation stimuli. Monitoring these molecules can provide a physiological read-out for inflammation. We present results from a multiparametric assays that used a combination of low-volume ELISA for secreted cytokines and automated cell imaging for cell surface markers to evaluate the effect of different mediators on inflammatory response with two cell models. First, human monocytic THP-1 cells were differentiated to macrophages and activated by LPS. Phenotypic changes in cell morphology were quantified by cell area and number of adherent cells using TL imaging. Secretion of IL-8, IL-1β and TNF-α was quantified by an automated low-volume ELISA (PuMA System). Second, primary human umbilical vein endothelial cells (HUVEC) were stimulated with inflammation cytokines (TNF-α, IFN-γ, and IL-1β), then PuMA System was used to quantify amount of MCP-1, IL-8, and IL-6 in cell supernatants. Expression of adhesion molecules VCAM and HLADR was quantified by measurement of total fluorescence from antibody-stained cells using an ImageXpress Pico system. Concentration dependent inhibition of inflammation by the compounds AG126 and MG132, SB202190 and PDTC were measured and EC50s calculated. Clear differences in cytokine expression were seen between the compounds consistent with reported mechanism of actions of the kinase inhibitors. The combination of imaging and microfluidic-based assays provides an efficient multiparametric assay system that can test the efficacy of anti-inflammatory compounds versus toxicity and provide insight into their MoA by selective inhibition of markers triggered by different signaling pathways.

High-speed cell recognition algorithm for ultrafast flow cytometer imaging system.

An optical time-stretch flow imaging system enables high-throughput examination of cells/particles with unprecedented high speed and resolution. A significant amount of raw image data is produced. A high-speed cell recognition algorithm is, therefore, highly demanded to analyze large amounts of data efficiently. A high-speed cell recognition algorithm consisting of two-stage cascaded detection and Gaussian mixture model (GMM) classification is proposed. The first stage of detection extracts cell regions. The second stage integrates distance transform and the watershed algorithm to separate clustered cells. Finally, the cells detected are classified by GMM. We compared the performance of our algorithm with support vector machine. Results show that our algorithm increases the running speed by over 150% without sacrificing the recognition accuracy. This algorithm provides a promising solution for high-throughput and automated cell imaging and classification in the ultrafast flow cytometer imaging platform.

Evidence Implicating Immunological Host Effects in the Efficacy of Metronomic Low-Dose Chemotherapy.

Conventional chemotherapy drugs administered at a maximum tolerated dose (MTD) remains the backbone for treating most cancers. Low-dose metronomic (LDM) chemotherapy, which utilizes lower, less toxic, doses given on a close regular basis over prolonged periods, is an alternative and better tolerated potential strategy to improve chemotherapy. LDM chemotherapy has been evaluated preclinically and clinically and has shown therapeutic benefit, in both early and advanced stage metastatic disease, especially when used as a maintenance therapy. However, knowledge about the antitumor mechanisms by which LDM chemotherapy acts remain limited. Here we characterized the effects of LDM and MTD capecitabine therapy on tumor and host cells using high-throughput systems approaches involving mass spectrometry flow cytometry and automated cell imaging followed by in vivo analyses of such therapies. An increase in myeloid and T regulatory cells and a decrease in NK and T cytotoxic cells were found in MTD-capecitabine-treated tumors compared with LDM-capecitbine-treated tumors. Plasma from MTD capecitabine-treated mice induced a more tumorigenic and metastatic profile in both breast and colon carcinoma cells than plasma from mice treated with LDM capecitabine. These results correlated, in part, with in vivo studies using models of human or mouse advanced metastatic disease, where the therapeutic advantage of MTD capecitabine was limited despite a substantial initial antitumor activity found in the primary tumor setting. Overall these results implicate a possible contribution of immunologic host effects in accounting for the therapeutic limitations of MTD compared with LDM capecitabine. Cancer Res; 76(20); 5983-93. ©2016 AACR.

A Simple yet Accurate Method for the Estimation of the Biovolume of Planktonic Microorganisms.

Determining the biomass of microbial plankton is central to the study of fluxes of energy and materials in aquatic ecosystems. This is typically accomplished by applying proper volume-to-carbon conversion factors to group-specific abundances and biovolumes. A critical step in this approach is the accurate estimation of biovolume from two-dimensional (2D) data such as those available through conventional microscopy techniques or flow-through imaging systems. This paper describes a simple yet accurate method for the assessment of the biovolume of planktonic microorganisms, which works with any image analysis system allowing for the measurement of linear distances and the estimation of the cross sectional area of an object from a 2D digital image. The proposed method is based on Archimedes’ principle about the relationship between the volume of a sphere and that of a cylinder in which the sphere is inscribed, plus a coefficient of ‘unellipticity’ introduced here. Validation and careful evaluation of the method are provided using a variety of approaches. The new method proved to be highly precise with all convex shapes characterised by approximate rotational symmetry, and combining it with an existing method specific for highly concave or branched shapes allows covering the great majority of cases with good reliability. Thanks to its accuracy, consistency, and low resources demand, the new method can conveniently be used in substitution of any extant method designed for convex shapes, and can readily be coupled with automated cell imaging technologies, including state-of-the-art flow-through imaging devices.

Differential Regulation of Progesterone Receptor-Mediated Transcription by CDK2 and DNA-PK.

Progesterone receptor (PR) function is altered by cell signaling, but the mechanisms of kinase-specific regulation are not well defined. To examine the role of cell signaling in the regulation of PR transcriptional activity, we have utilized a previously developed mammalian-based estrogen-response element promoter array cell model and automated cell imaging and analysis platform to visualize and quantify effects of specific kinases on different mechanistic steps of PR-mediated target gene activation. For these studies, we generated stable estrogen-response element array cell lines expressing inducible chimeric PR that contains a swap of the estrogen receptor-α DNA-binding domain for the DNA-binding domain of PR. We have focused on 2 kinases important for steroid receptor activity: cyclin-dependent kinase 2 and DNA-dependent protein kinase. Treatment with either a Cdk1/2 inhibitor (NU6102) or a DNA-dependent protein kinase inhibitor (NU7441) decreased hormone-mediated chromatin decondensation and transcriptional activity. Further, we observed a quantitative reduction in the hormone-mediated recruitment of select coregulator proteins with NU6102 that is not observed with NU7441. In parallel, we determined the effect of kinase inhibition on hormone-mediated induction of primary and mature transcripts of endogenous genes in T47D breast cancer cells. Treatment with NU6102 was much more effective than NU7441, in inhibiting induction of PR target genes that exhibit a rapid increase in primary transcript expression in response to hormone. Taken together, these results indicate that the 2 kinases regulate PR transcriptional activity by distinct mechanisms.