analysis-cancer-cell-invasion-anti-metastatic-drug-screening-using

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

Bar Ilan University

Bar-Ilan University

Direct quantitative experimental investigations of the function of lymphocytes and other immune cells are challenging due to the cell mobility and the complexity of intercellular communications. In order to facilitate such investigations, an in vitro system is required that is noninvasive and provides kinetic data on cellular responses to challenges such as drug treatments. The present work reports the development of a disposable, inexpensive polymer-made device, the Polymer Live Cell Array (PLCA), for real-time, kinetic analysis of immune cells. The PLCA proved to be optically and biologically compatible, thus individual immune cells can be observed and treated independently without being tethered. The cells share a common space which facilitates cellular communications via secreted molecules or via direct intercellular interactions. These properties facilitate real-time, non-intrusive, repeated measurements of immune cells under multiple experimental treatments.

Polymer live-cell array for real-time kinetic imaging of immune cells.

Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient.

The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: methodology.

Multicellular spheroid models have been recognized as superior to monolayer cell cultures in antitumor drug screening, but their commercial adaptation in the pharmaceutical industry has been delayed, primarily due to technological limitations. The current study presents a new spheroid culture platform that addresses these technical restrictions. The new culturing device is based on a multiwell plate equipped with a glass bottom patterned with an array of UV adhesive microchambers. Each microchamber is designed to accommodate a single spheroid. The system facilitates the simultaneous creation and culturing of a large number of spheroids, as well as screening their response to antitumor drugs. The volume of the spheroids is easily controlled by seeding density. The location of each spheroid is preserved in the same microchamber throughout its growth, treatment with soluble agents, and imaging. The growth ratio parameter, a non-intrusive size analysis of the same spheroid before and after exposure to drugs, was found to be a sensitive indicator for the reaction of MCF7 breast cancer spheroids to cytotoxic drugs. This feature helps reveal the heterogeneity within the spheroid population during the formation process and their drug response, and provides an opportunity to detect specific, highly active or drug-resistant spheroid sub-groups. The advantages of this spheroid-based system make it an efficient drug-screening tool that may be valuable to related fields of research and clinical applications.

A polymer microstructure array for the formation, culturing, and high throughput drug screening of breast cancer spheroids.

The cryopreservation and thawing processes are known to induce many deleterious effects in cells and might be detrimental to several cell types. There is an inherent variability in cellular responses among cell types and within individual cells of a given population with regard to their ability to endure the freezing and thawing process. The aim of this study was to evaluate the fate of cryopreserved cells within an optical cryo apparatus, the individual-cell-based cryo-chip (i3C), by monitoring several basic cellular functional activities at the resolution of individual cells.

The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. II: functional activity of cryopreserved cells.

Nitric oxide (NO) is recognized as one of the major immune system agents involved in the pathogenesis and control of various diseases that may benefit from novel drug development, by exploiting NO signaling pathways and targets. This calls for detection of both intracellular levels of NO and expression of its synthesizing enzymes (NOS) in individual, intact, living cells. Such measurements are challenging, however, due to short half-life, low and fluctuating concentrations of NO, cellular heterogeneity, and inability to trace the same cells over time. The current study presents a device and methodology for correlative analysis of NO generation rates and NOS levels in the same individual cells, utilizing fluorescent imaging followed by immunohistochemistry (IHC). U937 promonocyte cell populations demonstrated significant heterogeneity in their baseline levels, in NO-generation kinetics, and in their response rates to stimuli. Individual cell analysis exposed cell subgroups which showed enhanced NO production upon stimulation, concomitantly with significant up-regulation of inducible NOS (iNOS) levels. Exogenous NO modulated the expression of iNOS in nondifferentiated cells within 1 h, in a dose-dependent manner, while treatment with lysophosphatidylcholine (LPC) enhanced the expression of iNOS, demonstrating a nondependence on NO production.

Correlative analyses of nitric oxide generation rates and nitric oxide synthase levels in individual cells using a modular cell-retaining device.

Living cells are known to exhibit great morphological, functional, spatial and temporal heterogeneity. Hence, the study of cells in a bulk, whether this bulk is homogenous or heterogeneous, does not provide sufficiently detailed or interpretable results. An advantageous approach would rather be a comprehensive study of cell biological activity in single isolated living cells. In this study, we present an imaging approach for studying pre-apoptotic and very early apoptotic events, during cell death induced by lysophosphatidylcholine (LPC) at the single cell level. The aim of this study is to investigate intracellular events, such as the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) formation, before and immediately after LPC introduction to the lymphocytes at the level of individual cells. A new protocol of sequential staining was developed to study the relation between early apoptosis signs (PS externalization), MMP changes and intracellular ROS production rates at an individual Jurkat cell resolution. Simultaneous kinetic assessments of MMP, intracellular ROS levels and phosphatidylserine (PS) externalization were performed at a single cell resolution, using Optical LiveCell™ Array technology and image analysis. The parameters were measured and analyzed both before and during exposure to inducers in a Jurkat cell population, including three groups of single cells: spontaneous apoptotic cells, induced apoptotic cells and fully functional living cells. Exogenous LPC caused a heterogeneous intracellular response among Jurkat cells immediately after its introduction. Subgroups of cells with opposite changes of MMP and different kinetics of ROS increase, were revealed within the whole cell population. The subset of apoptosis-induced Jurkat cells, which became apoptotic within 3h after the LPC introduction, exhibited higher initial MMP compared to fully functional or spontaneous apoptotic cells. LPC-induced apoptosis was accompanied by a concomitant increase in intracellular ROS levels. In the present study, a method is described to assess the intracellular events in cells which were initially different in their physiological status. The individual T lymphocytes (Jurkat cells) in vitro have various susceptibilities to LPC effects at the very early stage of contact with the inducer. The apoptotic effect of LPC in individual Jurkat cells is associated with a relatively higher initial MMP before the introduction of the inducer and with a faster ROS formation within the affected cells. Such divergence may be significant in regulating the balance of lymphocyte subsets in pathological sites, either maintaining or preventing the inflammation components of atherosclerosis. We conclude that the presented approach provides the researcher, not only with the cell retaining methodology, but with opportunities to observe and find the distinctive cell subsets within the whole cell population as well, thus helping to define more exactly the role and importance of such sub-populations in physiological or pathological conditions.

The use of sequential staining for detection of heterogeneous intracellular response of individual Jurkat cells to lysophosphatidylcholine.

In order to study cell-cell variation with respect to enzymatic activity, individual live cell analysis should be complemented by measurement of single cell content in a biomimetic environment on a cellular scale arrangement. This is a challenging endeavor due to the small volume of a single cell, the low number of target molecules and cell motility. Micro-arrayed donut-shaped chambers (DSCs) of femtoliter (fL), picoliter (pL), and nanoliter (nL) volumes have been developed and produced for the analysis of biochemical reaction at the molecular, cellular and multicellular levels, respectively. DSCs are micro-arrayed, miniature vessels, in which each chamber acts as an individual isolated reaction compartment. Individual live cells can settle in the pL and nL DSCs, share the same space and be monitored under the microscope in a noninvasive, time-resolved manner. Following cell lysis and chamber sealing, invasive kinetic measurement based on cell content is achieved for the same individual cells. The fL chambers are used for the analysis of the same enzyme reaction at the molecular level. The various DSCs were used in this proof-of-principle work to analyze the reaction of intracellular esterase in both primary and cell line immune cell populations. These unique DSC arrays are easy to manufacture and offer an inexpensive and simple operating system for biochemical reaction measurement of numerous single cells used in various practical applications. 

Donut-shaped chambers for analysis of biochemical processes at the cellular and subcellular levels.

Cell populations represent intrinsically heterogeneous systems with a high level of spatiotemporal complexity. Monitoring and understanding cell-to-cell diversity is essential for the research and application of intra- and interpopulation variations. Optical analysis of live cells is challenging since both adherent and nonadherent cells change their spatial location. However, most currently available single-cell techniques do not facilitate treatment and monitoring of the same live cells over time throughout multistep experiments. An imaging-dish-based live cell array (ID-LCA) has been developed and produced for cell handling, culturing, and imaging of numerous live cells. The dish is composed of an array of pico scale cavities-pico wells (PWs) embossed on its glass bottom. Cells are seeded, cultured, treated, and spatiotemporally measured on the ID-LCA, while each cell or small group of cells are locally constrained in the PWs. Finally, predefined cells can be retrieved for further evaluation. Various types of ID-LCAs were used in this proof-of-principle work, to demonstrate on-ID-LCA transfection of fluorescently tagged chimeric proteins, as well as the detection and kinetic analysis of their induced translocation. High variability was evident within cell populations with regard to protein expression levels as well as the extent and dynamics of protein redistribution. The association of these parameters with cell morphology and functional parameters was examined. Both the new methodology and the device facilitate research of the translocation process at individual cell resolution within large populations and thus, can potentially be used in high-throughput fashion. 

Real-time quantification of protein expression and translocation at individual cell resolution using imaging-dish-based live cell array.

Estrogen-induced apoptosis has become a successful treatment for postmenopausal metastatic, estrogen receptor-positive breast cancer. Nitric oxide involvement in the response to this endocrine treatment and its influence upon estrogen receptor-positive breast cancer progression is still unclear. Nitric oxide impact on the MCF7 breast cancer line, before and after estrogen-induced apoptosis, was investigated in 3D culture systems using unique live-cell imaging methodologies. Spheroids were established from MCF7 cells vulnerable to estrogen-induced apoptosis, before and after exposure to estrogen. Spheroids derived from estrogen-treated cells exhibited extensive apoptosis levels with downregulation of estrogen receptor expression, low proliferation rate and reduced metabolic activity, unlike spheroids derived from non-treated cells. In addition to basic phenotypic differences, these two cell cluster types are diverse in their reactions to exogenous nitric oxide. A dual effect of nitric oxide was observed in the breast cancer phenotype sensitive to estrogen-induced apoptosis. Nitric oxide, at the nanomolar level, induced cell proliferation, high metabolic activity, downregulation of estrogen receptor and enhanced collective invasion, contributing to a more aggressive phenotype. Following hormone supplementation, breast cancer 3D clusters were rescued from estrogen-induced apoptosis by these low nitric oxide-donor concentrations, since nitric oxide attenuates cell death levels, upregulates survivin expression and increases metabolic activity. Higher nitric oxide concentrations (100nM) inhibited cell growth, metabolism and promoted apoptosis. These results suggest that nitric oxide, in nanomolar concentrations, may inhibit estrogen-induced apoptosis, playing a major role in hormonal therapy. Inhibiting nitric oxide activity may benefit breast cancer patients and ultimately reduce tumor recurrence.

Article	Year
Polymer live-cell array for real-time kinetic imaging of immune cells. Biomaterials\| PubMed ID: 20346503	2010
The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: methodology. BMC cell biology\| PubMed ID: 20609216	2010
A polymer microstructure array for the formation, culturing, and high throughput drug screening of breast cancer spheroids. Biomaterials\| PubMed ID: 20692698	2010
The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. II: functional activity of cryopreserved cells. BMC cell biology\| PubMed ID: 20973993	2010
Correlative analyses of nitric oxide generation rates and nitric oxide synthase levels in individual cells using a modular cell-retaining device. Analytical chemistry\| PubMed ID: 22839699	2012
The use of sequential staining for detection of heterogeneous intracellular response of individual Jurkat cells to lysophosphatidylcholine. Journal of immunological methods\| PubMed ID: 23063692	2013
Donut-shaped chambers for analysis of biochemical processes at the cellular and subcellular levels. Lab on a chip\| PubMed ID: 24829933	2014
Real-time quantification of protein expression and translocation at individual cell resolution using imaging-dish-based live cell array. Analytical and bioanalytical chemistry\| PubMed ID: 25258284	2014
Nitric oxide is cytoprotective to breast cancer spheroids vulnerable to estrogen-induced apoptosis. Oncotarget\| PubMed ID: 29312577	2017

Maria Sobolev

Biography

JoVE Journal Publications

Other Publications