Live Cell Samples Research Articles

Abstract P4-12-06: Quantification of HER2-driven signaling (HER2S) inhibition of four different anti-HER2 drugs tested ex vivo in live primary HER2-negative breast cancer cell samples with abnormal HER2 signaling activity

Abstract Background: A new functional cellular analysis platform, the CELx HER2 Signaling Profile (CELx HSP) Test, uses a label-free impedance biosensor to measure HER2 signaling activity in live tumor cells. A recently completed study quantified HER2-driven signaling activity in epithelial cell samples extracted and cultured from fresh breast tissue specimens obtained from 34 patients with HER2-negative breast cancer (DAKO 0 or 1+). Of the cell samples tested, 7 of 34 HER2-negative breast tumor patients (20.5%; 95% CI=10%-37%) were found to have abnormal HER2 signaling activity (HER2S+). The current study set out to: 1) evaluate the primary cells with abnormal HER2-driven signaling with four HER2 signal inhibitors - pertuzumab, lapatinib, neratinib, afatinib; and 2) evaluate the same four HER2 signal inhibitors with 9 HER2-positive cell lines. The objective was to quantify the percentage of HER2-driven signaling activity each drug could inhibit ex vivo in the primary cell samples and cell lines. Comparing the results between the HER2-negative primary cells and the HER2-positive cell lines was also of interest. The anti-HER2 drug, trastuzumab, was not studied because its primary mechanism of action does not appear to be direct mediation of HER2-driven signaling. Methods: Epithelial cells from the 7 HER2-negative tumor specimens with abnormal HER2-driven signaling (HER2S+) and the 9 HER2-positive cell lines were obtained. Real time live cell response to NRG1, a specific HER2/HER3 agonist, with or without a HER2 targeted drug (pertuzumab, lapatinib, neratinib, afatinib) was measured and quantified using an xCELLigence RTCA impedance biosensor (ACEA Biosciences, San Diego, CA). Clinically relevant concentrations of the HER2 drugs were used. From these responses, the percentage inhibition of the HER2-driven signaling initiated by NRG1 by the HER2 drugs was determined. Results: Each of the HER2 drugs inhibited an average of at least 69% of the HER2-driven signaling activated by NRG1 stimulation in the HER2-negative primary cell samples; the highest level of inhibition was found with the two irreversible covalent dual RTKi's, afatinib and neratinib. All of the HER2 drugs inhibited a greater percentage of HER2-driven signaling in the HER2-negative primary tumor cells than in the HER2-positive cell lines. table 1 Avg. % NRG1 Inhibition Cell LinesPrimariesHER2 DrugsMechanism of Action(HER2+)(HER2-)PertuzumabHER2 dimerization inhibitor46%78%LapatinibReversible Dual RTKi (HER2, EGF)15%69%AfatinibIrreversible Covalent Dual RTKi (HER2, EGF)47%93%NeratinibIrreversible Covalent Dual RTKi (HER2, EGF)95%100% Conclusions: These findings provide strong evidence that HER2 signal inhibitors are effective in blocking abnormal levels of HER2-driven signaling (HER2S+) ex vivo in live primary cells from breast cancer patients with normal expression levels of HER2. These results suggest a new group of breast cancer patients, HER2-negative with abnormal HER2 signaling (HER2-/HER2S+), may benefit from the addition of HER2 signal inhibitors to current combination therapeutic regimens. Additional studies to confirm these findings are underway. Citation Format: Laing L, Burns D, Huang Y, MacNeil I, Rich B, Myhre S, Soltani S, Sullivan B. Quantification of HER2-driven signaling (HER2S) inhibition of four different anti-HER2 drugs tested ex vivo in live primary HER2-negative breast cancer cell samples with abnormal HER2 signaling activity [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-06.

A Multi-Modality CMOS Sensor Array for Cell-Based Assay and Drug Screening.

In this paper, we present a fully integrated multi-modality CMOS cellular sensor array with four sensing modalities to characterize different cell physiological responses, including extracellular voltage recording, cellular impedance mapping, optical detection with shadow imaging and bioluminescence sensing, and thermal monitoring. The sensor array consists of nine parallel pixel groups and nine corresponding signal conditioning blocks. Each pixel group comprises one temperature sensor and 16 tri-modality sensor pixels, while each tri-modality sensor pixel can be independently configured for extracellular voltage recording, cellular impedance measurement (voltage excitation/current sensing), and optical detection. This sensor array supports multi-modality cellular sensing at the pixel level, which enables holistic cell characterization and joint-modality physiological monitoring on the same cellular sample with a pixel resolution of 80 μm × 100 μm. Comprehensive biological experiments with different living cell samples demonstrate the functionality and benefit of the proposed multi-modality sensing in cell-based assay and drug screening.

Quantitative differential phase contrast imaging in an LED array microscope

Illumination-based differential phase contrast (DPC) is a phase imaging method that uses a pair of images with asymmetric illumination patterns. Distinct from coherent techniques, DPC relies on spatially partially coherent light, providing 2× better lateral resolution, better optical sectioning and immunity to speckle noise. In this paper, we derive the 2D weak object transfer function (WOTF) and develop a quantitative phase reconstruction method that is robust to noise. The effect of spatial coherence is studied experimentally, and multiple-angle DPC is shown to provide improved frequency coverage for more stable phase recovery. Our method uses an LED array microscope to achieve real-time (10 Hz) quantitative phase imaging with in vitro live cell samples.

Ratiometric time-gated luminescence probe for hydrogen sulfide based on lanthanide complexes.

Developments of ratiometric bioprobes are highly appealing due to the superiority of their self-calibration capability for the quantitative biotracking. In this work, we designed and synthesized a novel lanthanide complex-based ratiometric luminescence probe, [4'-(2,4-dinitrophenyloxy)-2,2':6',2″-terpyridine-6,6″-diyl]bis(methylenenitrilo) tetrakis(acetate)-Eu(3+)/Tb(3+) (NPTTA-Eu(3+)/Tb(3+)), for the specific recognition and quantitative time-gated luminescence detection of hydrogen sulfide (H2S) in aqueous and living cell samples. Due to the presence of the photoinduced electron transfer (PET) process from the terpyridine-Eu(3+)/Tb(3+) moiety to 2,4-dinitrophenyl (DNP), the probe itself is weakly luminescent. In physiological pH aqueous media, the reaction of NPTTA-Eu(3+)/Tb(3+) with H2S leads to the cleavage of DNP moiety from the probe molecule, which affords the deprotonated (4'-hydroxy-2,2':6',2″-terpyridine-6,6″-diyl)bis(methylenenitrilo) tetrakis(acetate)-Eu(3+)/Tb(3+) and terminates the PET process. Meanwhile, the intensity of Tb(3+) emission at 540 nm is remarkably increased, while that of the Eu(3+) emission at 610 nm is slightly decreased. After the reaction, the intensity ratio of Tb(3+) emission to Eu(3+) emission, I540/I610, was ∼220-fold increased, and the dose-dependent enhancement of I540/I610 showed a good linearity upon the increase of H2S concentration with a detection limit of 3.5 nM. This unique luminescence response allowed NPTTA-Eu(3+)/Tb(3+) to be conveniently used as a ratiometric probe for the time-gated luminescence detection of H2S with I540/I610 as a signal. In addition, the applicability of the probe for the quantitative time-gated luminescence imaging of intracellular H2S in living cells was investigated. The results demonstrated the efficacy and advantage of the new probe for the time-gated luminescence cell imaging application.

A Lanthanide Complex-Based Ratiometric Luminescence Probe for Time-Gated Luminescence Detection of Intracellular Thiols

A lanthanide complex-based ratiometric luminescence probe, [4'-(2,4-dinitrobenzenesulfonyloxy)-2,2':6',2''-terpyridine-6,6''-diyl] bis(methylenenitrilo) tetrakis(acetate)-Eu(3+)/Tb(3+) (NSTTA-Eu(3+)/Tb(3+)), has been designed and synthesized for the specific recognition and time-gated luminescence detection of biothiols in physiological pH aqueous media. The probe itself is almost nonluminescent due to the presence of photoinduced electron transfer (PET) from the terpyridine-Ln(3+) moiety to the 2,4-dinitrobenzenesulfonyl (DNBS) moiety. In the presence of biothiols, the reaction of NSTTA-Eu(3+)/Tb(3+) with biothiols results in the cleavage of DNBS to afford the deprotonated (4'-hydroxy-2,2':6',2''-terpyridine-6,6''-diyl) bis(methylenenitrilo) tetrakis(acetate)-Eu(3+)/Tb(3+) (HTTA-Eu(3+)/Tb(3+)), which terminates the PET process. After the reaction, the intensity of Eu(3+) emission at 610 nm is unchanged, while that of Tb(3+) emission at 540 nm is remarkably increased, which provides a ~36-fold enhanced intensity ratio of Tb(3+) emission to Eu(3+) emission (I540/I610). This unique luminescence response allows NSTTA-Eu(3+)/Tb(3+) to be used as a ratiometric probe for the time-gated luminescence detection of biothiols, using the intensity ratio of I540/I610 as a signal. Thus, based on the probe NSTTA-Eu(3+)/Tb(3+), a ratiometric time-gated luminescence detection method for biothiols was established and successfully used for the quantitative detection of the total biothiols in several living cell samples.

Development of a Ruthenium(II) Complex-Based Luminescent Probe for Hypochlorous Acid in Living Cells

A novel Ru(II) complex, [Ru(bpy)2(DNPS-bpy)](PF6)2 (bpy: 2,2'-bipyridine, DNPS-bpy: 4-(2,4-dinitrophenylthio)methylene-4'-methyl-2,2'-bipyridine), has been designed and synthesized as a highly sensitive and selective luminescence probe for the recognition and detection of hypochlorous acid (HOCl) in living cells by exploiting a "signaling moiety-recognition linker-quencher" sandwich approach. The complex possesses large stokes shift (170 nm), long emission wavelength (626 nm), and low cytotoxicity. Owing to the effective photoinduced electron transfer (PET) from Ru(II) center to the electron acceptor, 2,4-dinitrophenyl (DNP), the red-emission of bipyridine-Ru(II) complex was completely withheld. In aqueous media, HOCl can trigger an oxidation reaction to cleave the DNP moiety from the Ru(II) complex, which results in the formation of a highly luminescent bipyridine-Ru(II) complex derivative, [Ru(bpy)2(COOH-bpy)](PF6)2 (COOH-bpy: 4'-methyl-2,2'-bipyridyl-4-carboxylic acid), accompanied by a 190-fold luminescence enhancement. Cell imaging experimental results demonstrated that [Ru(bpy)2(DNPS-bpy)](PF6)2 is membrane permeable, and can be applied for capturing and visualizing the exogenous/endogenous HOCl molecules in living cell samples. The development of this Ru(II) complex probe not only provides a useful tool for monitoring HOCl in living systems, but also strengthens the application of transition metal complex-based luminescent probes for bioimaging.

In vivo MRI discrimination between live and lysed iron-labelled cells using balanced steady state free precession

The goal of this study was to evaluate the ability of balanced steady state free precession (b-SSFP) magnetic resonance imaging sequence to distinguish between live and lysed iron-labelled cells. Human breast cancer cells were labelled with iron oxide nanoparticles. Cells were lysed using sonication. Imaging was performed at 3 T. The timing parameters for b-SSFP and the number of iron-labelled cells in samples were varied to optimise the b-SSFP signal difference between live and lysed iron-labelled cell samples. For in vivo experiments, cells were mixed with Matrigel and implanted into nude mice. Three mice implanted with live labelled cancer cells were irradiated to validate this method. Lysed iron-labelled cells have a significantly higher signal compared with live, intact iron-labelled cells in bSSFP images. The contrast between live and dead cells can be maximised by careful optimisation of timing parameters. A change in the b-SSFP signal was measured 6 days after irradiation, reflecting cell death in vivo. Histology confirmed the presence of dead cells in the implant. Our results show that the b-SSFP sequence can be optimised to allow for the discrimination of live iron-labelled cells and lysed iron-labelled cells in vitro and in vivo.

Dark-field optical coherence microscopy

Dark-field illumination is known to enhance scattering contrast in optical microscopy. We combined this concept with Fourier domain optical coherence microscopy (OCM). The detection and illumination paths are decoupled, and only the scattered light originating from the sample generates the tomogram signal, whereas any specular reflection is highly suppressed. We analyze and discuss this dark-field OCM concept and present its superior imaging quality on live cell samples.

Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells.

Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications.

Monitoring changes of cellular metabolism and microviscosity <italic>in vitro</italic> based on time-resolved endogenous fluorescence and its anisotropy decay dynamics

Reduced nicotinamide adenine dinucleotide (NADH) is a well-known metabolic coenzyme and endogenous fluorophore. In this study, we develop a system that simultaneously measures time- and wavelength-resolved fluorescence to extract free and protein-bound NADH signals from total cellular fluorescence. We analyze temporal characteristics of NADH fluorescence in a mixture of NADH and lactate dehydrogenase (LDH) as well as in living cell samples. The results show that in both the NADH/LDH mixture and cell samples, a fraction of free NADH and protein-bound components can be identified. The extracted free and bound NADH signals are confirmed by time-resolved measurement of anisotropy decay of NADH fluorescence, based on the fact that free NADH is a small fluorescent molecule with much shorter rotational diffusion time than bound NADH. The ratio of free NADH signal to bound NADH signal is very different between normal and cancer cervical epithelial cells. In addition, the ratio changes significantly when the cell samples are treated with a mitochondrial inhibitor or uncoupler, demonstrating that the method is sensitive to monitor cellular metabolic activity. Finally, we demonstrate that the microviscosity for relatively small molecules such as NADH in cells could be extracted from wavelength- and time-resolved NADH fluorescence of living cell samples.

Mouse Fibroblast Cell Adhesion Studied by Neutron Reflectometry

Neutron reflectometry (NR) was used to examine live mouse fibroblast cells adherent on a quartz substrate in a deuterated phosphate-buffered saline environment at room temperature. These measurements represent the first, to our knowledge, successful visualization and quantization of the interface between live cells and a substrate with subnanometer resolution using NR. NR data, attributable to the adhesion of live cells, were observed and compared with data from pure growth medium. Independently of surface cell density, the average distance between the center of the cell membrane region and the quartz substrate was determined to be ∼180 Å. The membrane region (∼80 Å thick) contains the membranes of cells that are inhomogeneously distributed or undulating, likely conforming to the nonplanar geometry of the supporting adherence proteins. A second region of cell membranes at a greater distance from the substrate was not detectable by NR due to the resolution limits of the technique employed. Attachment of the live cell samples was confirmed by interaction with both distilled water and trypsin. Distinct changes in the NR data after exposure indicate the removal of cells from the substrate.

A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission

Fluorescence detection of acceptor molecules sensitized by Förster resonance energy transfer (FRET) is a powerful method to study protein interactions in living cells. The method requires correction for donor spectral bleed-through and acceptor cross-excitation as well as the correct normalization of signals to account for varying fluorophore concentrations and imaging parameters. In this paper, we review different methods for FRET signal normalization and then present a rigorous model for sensitized emission measurements, which is both intuitive to understand and practical to apply. The method is validated by comparison with the acceptor photobleaching and donor lifetime-imaging techniques in live cell samples containing EYFP and ECFP tandem constructs exhibiting known amounts of FRET. By varying the stoichiometry of interaction in a controlled fashion, we show that information on the fractions of interacting donors and acceptors can be recovered. Furthermore, the method is tested by performing measurements on different microscopy platforms in both widefield and confocal imaging modes to show that signals recovered under different imaging conditions are in quantitative agreement. Finally, the method is applied in the study of dynamic interactions in the cyclin–cdk family of proteins in live cells. By normalizing the obtained signals for both acceptor and donor concentrations and using a FRET exhibiting control construct for calibration, stoichiometric changes in these interactions could be visualized in real time. The paper is written to be of practical use to researchers interested in performing sensitized emission measurements. The correct interpretation of the retrieved signals in a biological context is emphasized, and guidelines are given for the practical application of the developed algorithms.

Laser-assisted microdissection: a new tool for aquatic molecular parasitology

Laser-assisted microdissection (LMD) has been developed to isolate distinct cell populations from heterogeneous tissue sections, cytological preparations, or live cell samples. Downstream applications typically include gene expression studies using real-time PCR and array platforms, diagnostic PCR, and protein expression studies. LMD techniques are now commonplace in mainstream biological research and clearly have suitable applications in the field of aquatic pathology and parasitology. The present study used LMD to isolate 2 dinoflagellate parasites (Hematodinium spp.) from formalin-fixed paraffin-embedded tissue sections from 2 crustacean hosts, Cancer pagurus and Portunus trituberculatus. DNA was isolated from LMD parasite preparations, and partial regions (up to 300 bp) of the small subunit and the first internal transcribed spacer region of the rRNA gene complex from the Hematodinium spp. were PCR amplified using diagnostic primers. The amplification products were sequenced to confirm the identity of the targeted regions. The techniques, applications, and limitations of LMD to address questions in aquatic molecular pathology and parasitology are discussed.

Quantitative phase microscopy with asynchronous digital holography

We demonstrate a new method of measuring quantitative phase in imaging of biological materials. This method, asynchronous digital holography, employs knowledge of a moving fringe created by acousto-optic modulators to execute phase-shifting interferometry using two near-simultaneous interferograms. The method can be used to obtain quantitative phase images of dynamic biological samples on millisecond time scales. We present results on a standard sample, and on live cell samples.

Scanning cytometry with a LEAP: Laser‐enabled analysis and processing of live cells in situ

Scanning cytometry now has many of the features (and power) of multiparameter flow cytometry while keeping its own advantages as an imaging technology. Modern instruments combine capabilities of scanning cytometry with the ability to manipulate cells. A new technology, called LEAP (laser-enabled analysis and processing), offers a unique combination of capabilities in cell purification and selective macromolecule delivery (optoinjection). LEAP-mediated cell purification and optoinjection effects were assessed in model experiments using adherent and suspension cell types and cell mixtures plated and processed at different densities. Optoinjection effects were visualized by delivering fluorescent dextrans into cells. Results were analyzed using the LEAP instrument's own imaging system as well as by fluorescence and confocal microscopy. Live cell samples (adherent and suspension) could be purified to 90-100% purity with 50-90% yield, causing minimal cell damage depending on the cell type and plating density. Nearly one hundred percent of the targeted cells of all cell types examined could be successfully optoinjected with dextrans of 3-70 kDa, causing no visual damage to the cells. Indirect optoinjection effects were observed on untargeted cells within 5-60 microm to targeted areas under conditions used here. LEAP provides solutions in cell purification and targeted macromolecule delivery for traditional and challenging applications where other methods fall short.

Nano-probing of the membrane dynamics of rat pheochromocytoma by near-field optics

High-resolution analysis of activities of live cells is limited by the use of non-invasive methods. Apparatuses such as SEM, STM or AFM are not practicable because the necessary treatment or the harsh contact with system probe will disturb or destroy the cell. Optical methods are purely non-invasive, but they are usually diffraction limited and then their resolution is limited to approximately 1 μm. To overcome these restrictions, we introduce here the study of membrane activity of a live cell sample using a Scanning Near-field Optical Microscope (SNOM). A near field optical microscope is able to detect tiny vertical movement on the cell membrane in the range of only 1 nm or less, about 3 orders of magnitude better than conventional optical microscopes. It is a purely non-invasive, non-contact method, so the natural life activity of the sample is unperturbed. In this report, we demonstrated the nanometer-level resolving ability of our SNOM system analyzing cardiomyocytes samples of which membrane movement is known, and then we present new intriguing data of sharp 40 nm cell membrane sudden events on rat pheochromocytoma cell line PC12. All the measurements are carried out in culture medium with alive and unperturbed samples. We believe that this methodology will open a new approach to investigate live samples. The extreme sensitivity of SNOM allows measurements that are not possible with any other method on live biomaterial paving the way for a broad range of novel studies and applications.