Dielectrophoresis-based cellular microarray chip for anticancer drug screening in perfusion microenvironments

We present a dielectrophoresis (DEP)-based cellular microarray chip for cell-based anticancer drug screening in perfusion microenvironments. Human breast cancer cells, MCF7, were seeded into the chip and patterned via DEP forces onto the planar interdigitated ring electrode (PIRE) arrays. Roughly, only one third of the cell amount was required for the chip compared to that for a 96-well plate control. Drug concentrations (cisplatin or docetaxel) were stably generated by functional integration of a concentration gradient generator (CGG) and an anti-crosstalk valve (ACV) to treat cells for 24 hours. Cell viability was quantified using a dual staining method. Results of cell patterning show substantial uniformity of patterned cells (92 ± 5 cells per PIRE). Furthermore, after 24 hour drug perfusion, no statistical significance in dose-responses between the chip and the 96-well plate controls was found. The IC(50) value from the chip also concurred with the values from the literature. Moreover, the perfusion culture exhibited reproducibility of drug responses of cells on different PIREs in the same chamber. The chip would enable applications where cells are of limited supply, and supplement microfluidic perfusion cultures for clinical practices.     

Microfluidic platform for studying the anti-cancer effect of ursolic acid on tumor spheroid

At present, the probability that a new anti-tumor drug will eventually succeed in clinical trials is extremely low. In order to make up for this shortcoming, the use of a three-dimensional (3D) cell culture model for secondary screening is often necessary. Cell spheroid is the easiest 3D model tool for drug screening. In this study, the microfluidic chip with a microwell array was manufactured, which could allow the formation of tumor spheroids with uniform size and easily retrieve cell spheroids from the chip. Cell spheroids were successfully cultured for over 15 days and the survival rate was as high as 80%. Subsequently, cellular response to the ursolic acid (UA) was observed on the chip. Compared to the monolayer culture cells in vitro, the tumor spheroids showed minor levels of epithelial-mesenchymal transition fluctuation after drug treatment. The mechanism of cell spheroid resistance to UA was further verified by detecting the expression level of upstream pathway proteins. But the invasive ability of tumor spheroids was attenuated when the duration of action of UA extended. The anti-cancer effect of UA was innovatively evaluated on breast cancer by using the microfluidic device, which could provide a basis and direction for future preclinical research on UA.     

Inflammatory mimetic microfluidic chip by immobilization of cell adhesion molecules for T cell adhesion

Leukocyte adhesion to adhesion molecules on endothelial cells is important in immune function, cancer metastasis and inflammation. This cell-cell binding is mediated via cell adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) found on endothelial cells. Because these adhesion molecules on endothelial cells vary significantly across several disease conditions such as autoimmune diseases, inflammation or cancer metastasis, investigations of therapeutic agents that down-regulate leukocyte-endothelial interactions have been based on in vitro models using endothelial cell lines. Here we report a new model, an inflammatory mimetic microfluidic chip, which emulates leukocyte binding to cell adhesion molecules (CAM) by controlling the types and ratio of adhesion molecules. In our model, E-selectin was essential for the synergic binding of Jurkat T cells. Immunosuppressive drugs, such as tacrolimus (FK506) and cyclosporine A (CsA), were used to inhibit T cell interactions under the physiologic model of T cell migration at a ratio of 5?:?4.3?:?3.9 (E-selectin?:?ICAM-1?:?VCAM-1). Our results support the potential usefulness of the inflammatory mimetic microfluidic chip as a T cell adhesion assay tool with modified adhesion molecules for applications such as immunosuppressive drug screening. The inflammatory mimetic microfluidic chip can also be used as a biosensor in clinical diagnostics, drug efficacy tests and high throughput drug screening due to the dynamic monitoring capability of the microfluidic chip.     

Integration of intra- and extravasation in one cell-based microfluidic chip for the study of cancer metastasis

Most studies of cancer metastasis focus on cancer cell invasion utilizing adhesion assays that are performed independently, and are thus limited in their ability to mimic complex cancer metastasis on a chip. Here we report the development of an integrated cell-based microfluidic chip for intra- and extravasation that combines two assays on one chip for the study of the complex cascade of cancer metastasis. This device consists of two parts; one is an intravasation chamber for the three-dimensional (3-D) culture of cancer cells using a Matrigel matrix, and the other is an extravasation chamber for the detection of metastasized cancer cells by adhesion molecules expressed by epithelial cells. In this novel system, the intravasation and extravasation processes of cancer metastasis can be studied simultaneously using four screw valves. Metastatic LOVO and non-metastatic SW480 cells were used in this study, and the invasion of LOVOs was found to be higher compared to SW480. In contrast, invasion of cells treated with metalloproteinase (MMP) inhibitors decreased within the intravasation chamber. Degraded cancer cells from the intravasation chamber were detected within the extravasation chamber under physiological conditions of shear stress, and differences in binding efficiency were also detected when CA19-9 antibody, an inhibitor of cancer cell adhesion, was used to treat degraded cancer cells. Our results support the potential usefulness of this new 3D cell-based microfluidic system as a drug screening tool to select targets for the development of new drugs and to verify their effectiveness.     

Microfluidic three-dimensional biomimetic tumor model for studying breast cancer cell migration and invasion in the presence of interstitial flow

Cell migration and invasion are critical steps in cancer metastasis, which are the major cause of death in cancer patients. Tumor-associated macrophages(TAMs) and interstitial flow(IF) are two important biochemical and biomechanical cues in tumor microenvironment, play essential roles in tumor progression. However, their combined effects on tumor cell migration and invasion as well as molecular mechanism remains largely unknown. In this work, we developed a microfluidic-based 3 D breast cancer model by co-culturing tumor aggregates, macrophages, monocytes and endothelial cells within 3 D extracellular matrix in the presence of IF to study tumor cell migration and invasion. On the established platform, we can precisely control the parameters related to tumor microenvironment and observe cellular responses and interactions in real-time. When co-culture of U937 with human umbilical vein endothelial cells(HUVECs) or MDA-MB-231 cells and tri-culture of U937 with HUVECs and MDA-MB-231 cells, we found that mesenchymal-like MDA-MB-231 aggregates activated the monocytes to TAM-like phenotype macrophages. MDA-MB-231 cells and IF simultaneously enhanced the macrophages activation by the stimulation of colony-stimulating factor 1(CSF-1). The activated macrophages and IF further promoted vascular sprouting via vascular endothelial growth factor(VEGFα) signal and tumor cell invasion. This is the first attempt to study the interaction between macrophages and breast cancer cells under IF condition. Taken together, our results provide a new insight to reveal the important physiological and pathological processes of macrophages-tumor communication. Moreover, our established platform with a more mimetic 3 D breast cancer model has the potential for drug screening with more accurate results.     

基于微流控芯片的乳腺癌细胞微环境酸化检测

建立了基于微流控芯片的乳腺癌微环境酸化模型和动态检测微环境酸化情况的分析方法。设计了一种多层复合式微流控芯片,将乳腺癌细胞悬液引入含有水凝胶前体的芯片培养室后,在硝酸纤维素薄膜上固化形成3D培养支架。芯片通道连续灌流模拟血流供应,并将非电化学的pH检测器引入芯片,通过图像分析得到实时的pH变化。通过观察癌细胞的存活率、增殖率、乳酸水平及pH值,分析微环境的酸化情况,同时与正常细胞进行比较。结果表明,连续灌流培养7 d,乳腺癌细胞的存活率保持在90%以上;随着培养天数的增加,芯片上癌细胞微环境的pH值逐渐降低,且灌流速度越低,pH值下降越明显,而正常细胞微环境的pH值无明显变化。基于微流控芯片的微环境酸化检测平台可实时动态检测微环境的pH值,有望成为相关肿瘤研究的有力工具。     

Probing metabolic alterations in breast cancer in response to molecular inhibitors with Raman spectroscopy and validated with mass spectrometry

Rapid and accurate response to targeted therapies is critical to differentiate tumors that are resistant to treatment early in the regimen. In this work, we demonstrate a rapid, noninvasive, and label-free approach to evaluate treatment response to molecular inhibitors in breast cancer (BC) cells with Raman spectroscopy (RS). Metabolic reprogramming in BC was probed with RS and multivariate analysis was applied to classify the cells into responsive or nonresponsive groups as a function of drug dosage, drug type, and cell type. Metabolites identified with RS were then validated with mass spectrometry (MS). We treated triple-negative BC cells with Trametinib, an inhibitor of the extracellular-signal-regulated kinase (ERK) pathway. Changes measured with both RS and MS corresponding to membrane phospholipids, amino acids, lipids and fatty acids indicated that these BC cells were responsive to treatment. Comparatively, minimal metabolic changes were observed post-treatment with Alpelisib, an inhibitor of the mammalian target of rapamycin (mTOR) pathway, indicating treatment resistance. These findings were corroborated with cell viability assay and immunoblotting. We also showed estrogen receptor-positive MCF-7 cells were nonresponsive to Trametinib with minimal metabolic and viability changes. Our findings support that oncometabolites identified with RS will ultimately enable rapid drug screening in patients ensuring patients receive the most effective treatment at the earliest time point.

Rapid and accurate response to targeted therapies is critical to differentiate tumors that are resistant to treatment early in the regimen.     

Stimulated Raman scattering microscopy with spectral phasor analysis: applications in assessing drug–cell interactions

Statins have displayed significant, although heterogeneous, anti-tumour activity in breast cancer disease progression and recurrence. They offer promise as a class of drugs, normally used for cardiovascular disease control, that could have a significant impact on the treatment of cancer. Understanding their mode of action and accurately assessing their efficacy on live cancer cells is an important and significant challenge. Stimulated Raman scattering (SRS) microscopy is a powerful, label-free imaging technique that can rapidly characterise the biochemical responses of live cell populations following drug treatment. Here, we demonstrate multi-wavelength SRS imaging together with spectral phasor analysis to characterise a panel of breast cancer cell lines (MCF-7, SK-BR-3 and MDA-MB-231 cells) treated with two clinically relevant statins, atorvastatin and rosuvastatin. Label-free SRS imaging within the high wavenumber region of the Raman spectrum (2800–3050 cm⁻¹) revealed the lipid droplet distribution throughout populations of live breast cancer cells using biocompatible imaging conditions. A spectral phasor analysis of the hyperspectral dataset enables rapid differentiation of discrete cellular compartments based on their intrinsic SRS characteristics. Applying the spectral phasor method to studying statin treated cells identified a lipid accumulating phenotype in cell populations which displayed the lowest sensitivity to statin treatment, whilst a weaker lipid accumulating phenotype was associated with a potent reduction in cell viability. This study provides an insight into potential resistance mechanisms of specific cancer cells towards treatment with statins. Label-free SRS imaging provides a novel and innovative technique for phenotypic assessment of drug-induced effects across different cellular populations and enables effective analysis of drug–cell interactions at the subcellular scale.

Stimulated Raman scattering microscopy with spectral phasor analysis provides a label-free approach for phenotypic evaluation of drug-induced effects.     

Simple Method in Diagnosing Cancer Cells by a Novel Fluorescence Probe BMVC

Different cellular accumulations with distinct fluorescence properties of BMVC in cancer cells from normal cells allow us to establish a simple and economic method for the diagnosis of cancer cells. With using a light emitting diode to excite the BMVC molecule, microarray fluorescence analysis of a cell‐based glass chip provides an easy method towards the detection of a limited number of cancer cells.     

微流控芯片药物诱导细胞凋亡

发展了一种以微流控芯片为平台的药物诱导细胞凋亡的新方法.选择HeLa细胞为对象,通过浓度梯度芯片形成稳定的药物浓度梯度,诱导HeLa细胞凋亡,利用荧光能量共振转移（fluorescence resonance energy transfer,FRET）成像系统进行实时监测,分析细胞对不同浓度化合物的毒性反应.结果表明,细胞在顺铂诱导下发生明显的起泡和皱缩,FRET比率值逐渐降低,在药物浓度梯度作用下,芯片每个通道内细胞呈现不同程度的凋亡.该方法实现了药物浓度梯度诱导细胞凋亡的实时监测和定量分析,为抗肿瘤药物评价和高通量药物筛选提供了新的手段.     

Comprehensive two‐dimensional PC‐3 prostate cancer cell membrane chromatography for screening anti‐tumor components from Radix Sophorae flavescentis

Radix Sophorae flavescentis is generally used for the treatment of different stages of prostate cancer in China. It has ideal effects when combined with surgical treatment and chemotherapy. However, its active components are still ambiguous. We devised a comprehensive two‐dimensional PC‐3 prostate cancer cell membrane chromatography system for screening anti‐prostate cancer components in Radix Sophorae flavescentis . Gefitinib and dexamethasone were chosen as positive and negative drugs respectively for validation and optimization the selectivity and suitability of the comprehensive two‐dimensional chromatographic system. Five compounds, sophocarpine, matrine, oxymatrine, oxysophocarpine, and xanthohumol were found to have significant retention behaviors on the PC‐3 cell membrane chromatography and were unambiguously identified by time‐of‐flight mass spectrometry. Cell proliferation and apoptosis assays confirmed that all five compounds had anti‐prostate cancer effects. Matrine and xanthohumol had good inhibitory effects, with half maximal inhibitory concentration values of 0.893 and 0.137 mg/mL, respectively. Our comprehensive two‐dimensional PC‐3 prostate cancer cell membrane chromatographic system promotes the efficient recognition and rapid analysis of drug candidates, and it will be practical for the discovery of prostate cancer drugs from complex traditional Chinese medicines.     

Anticancer activities of manganese-based photoactivatable CO-releasing complexes (PhotoCORMs) with benzimidazole derivative ligands

Carbon monoxide is an important signaling molecule which is produced by heme oxygenase-1. CO shows antiproliferative activity against cancer cells; hence, activation of HO-1 is a significant inhibition strategy against tumor formation and survival of cancer cells. In this work, manganese-based CO-releasing molecules (CORMs) were designed and synthesized to inhibit breast cancer cell proliferation. Human invasive ductal breast cancer cells (MCF-7) were treated with the synthesized CORMs to investigate the effect of the complexes on breast cancer survival under UV light. In vitro experiments indicated that the complexes inhibited breast cancer cell proliferation, and further, the antiproliferative effects were increased under UV light. Thus, these novel CORMs may provide a drug template for the treatment of invasive ductal breast cancer.     

Resveratrol-induced cell inhibition of growth and apoptosis in MCF7 human breast cancer cells are associated with modulation of phosphorylated akt and caspase-9

Resveratrol (trans-3,4N,-5-trihydroxystilbene), a phytoalexin present in grapes and red wine, is emerging as a natural compound with potential anticancer properties. Here we show that resveratrol affects the growth of human breast cancer cell lines MCF7, MDA-MB-231, SK-BR-3, and Bcap-37 in a dose-dependent manner and that MCF7 is the most sensitive among the four cell lines. MCF7 cells treated with resveratrol showed typical characteristics of apoptosis including the poly (ADP-ribose) polymerase cleavage, TdT-mediated dUTP nick end labeling-positive staining, and morphologic changes. Phosphorylation of the oncogene product Akt was significantly reduced followed by decreased phosphorylation and increased processing of pro-caspase-9 on resveratrol treatment. These results indicate that resveratrol seems to exert its growth-inhibitory/apoptotic effect on the breast cancer cell line MCF7 via the Akt-caspase-9 pathway.     

Design of parallel microfluidic gradient-generating networks for studying cellular response to chemical stimuli

A microfluidic chip featuring laminar flow-based parallel gradient-generating networks was designed and fabricated. The microchip contains 5 gradient generators and 30 cell chambers where the resulting concentration gradients of drugs are delivered to stimulate on-chip cultured cells. The microfluidics exploits the advantage of lab-on-a-chip technology by integrating the generation of drug concentration gradients and a series of cell operations including seeding, culture, stimulation and staining into a chip. The microfluidic network was patterned on a glass wafer, which was further bonded to a PDMS film. A series of weir structures were fabricated on the cell culture reservoir to facilitate cell positioning and seeding. Cell injection and fluid delivery were controlled by a syringe pump. Steady parallel concentration gradients were generated by flowing two fluids in each network. Over time observation shows that the microchip was suitable for cell seeding and culture. The microchip described above was applied in studying the role of reduced glutathione (GSH) in mediating chemotherapy sensitivity of MCF-7 cells. MCF-7 cells were treated with concentration gradients of As₂O₃ and N-acetyl cysteine (NAC) for GSH modulation, followed by exposure to adriamycin. GSH levels were down-regulated upon As₂O₃ treatment and up-regulated upon NAC treatment. Suppression of intracellular GSH by treatment with As₂O₃ has been shown to increase sensitivity to adriamycin. Conversely, elevation of intracellular GSH by treatment with NAC leads to increased drug resistance. The integrated microfluidic chip is able to perform multiparametric pharmacological profiling with easy operation, and thus holds great potential for extrapolation to the cell based high-content drug screening. __________ Translated from Chinese Journal of Analytical Chemistry, 2008, 36(2): 143–149     

Stem cell membrane vesicle–coated nanoparticles for efficient tumor‐targeted therapy of orthotopic breast cancer

Nanoparticles‐based drug delivery strategies have been widely researched for cancer therapy. However, most of them are expected to accumulate in tumor sites via the enhanced permeability and retention (EPR) effect, which is insufficient to deliver the loaded drug into tumors. Cell membrane–camouflaged nanoparticles have obtained much attention for their excellent stability and long blood circulation and reduced the macrophage cells uptake in drug delivery. Herein, bone marrow–derived mesenchymal stem cell membrane vesicle (SCV)–coated paclitaxel (PTX)–loaded poly (lactide‐co‐glycolide) (PLGA) nanoparticles (SCV/PLGA/PTX) were fabricated as the efficient orthotopic breast cancer–targeted drug delivery system. The SCV/PLGA/PTX showed excellent stability, more controlled PTX release, and more effective antitumor effect in vitro. After administration in vivo, SCV/PLGA/PTX exhibited the long‐term retention and enhanced accumulation at tumor sites due to the immune escape and mesenchymal stem cell–mimicking cancer‐targeting capacity. As expected, the SCV/PLGA/PTX could significantly suppress the primary tumor growth by increased apoptosis and necrosis areas within tumor tissues and attenuated the toxic side effects of PTX in 4T1 orthotopic breast cancer model. The study indicated the mesenchymal stem cell membrane coating strategy was highly efficient for targeted drug delivery, which provided a new insight for precise and effective breast cancer treatment.     

A comparison of breast cancer tumor cells with varying expression of the Her2/neu receptor by Raman microspectroscopic imaging

The Her2/neu proto-oncogene is amplified in 25 to 30 percent of human primary breast carcinomas. The roles of Her2/neu have been reported before in literature, showing different relations to intracellular lipid composition. Here, we use Raman microspectroscopic imaging to reveal the chemical composition of single live cells from breast carcinoma cell lines MDA-MB-231, MDA-MB-435s and SK-BR-3, which express Her2/neu receptor in different extent. Average Raman spectra of the different cell populations show prominent lipid presence in all cell lines. With high significance, Raman difference spectra reveal increased lipid contents, as well as a lower degree of fatty acid saturation in the MDA-MB cell lines with respect to the SK-BR-3 cells. These results are confirmed by hierarchical cluster analysis of single cells. High internal consistency of the chemical compositions in the cell lines is shown by hierarchical cluster analysis on a single matrix composed of the data of different cells from a single cell line. Although Her2/neu expression is highest for SK-BR-3 cells, their lipid contents are lower than that of the MDA-MB cell lines, which express less to no Her2/neu receptors. Rather than metabolic rate or senescence, the degree of metastaticity of the cells appears to be related to the polyunsaturated fatty acid contents of the cells.     

Constructing real-time,wash-free,and reiterative sensors for cell surface proteins using binding-induced dynamic DNA assembly

Cell surface proteins are an important class of biomarkers for fundamental biological research and for disease diagnostics and treatment. In this communication, we report a universal strategy to construct sensors that can achieve rapid imaging of cell surface proteins without any separation by using binding-induced dynamic DNA assembly. As a proof-of-principle, we developed a real-time and wash-free sensor for an important breast cancer biomarker, human epidermal growth factor receptor-2 (HER2). We then demonstrated that this sensor could be used for imaging and sensing HER2 on both fixed and live breast cancer cells. Additionally, we have also incorporated toehold-mediated DNA strand displacement reactions into the HER2 sensor, which allows for reiterating (switching on/off) fluorescence signals for HER2 from breast cancer cells in real-time.     

基于微流控技术的细胞水平高通量药物筛选系统的研究进展

药物筛选是新药研发的关键步骤,创新药物的发现需要采用适当的药物作用靶点对大量化合物样品进行筛选。高通量筛选系统能够实现数千个反应同时测试和分析,大大提高了药物筛选的实验规模和效率。其中基于细胞水平的高通量药物筛选系统因为更加接近人体生理条件,成为主要的筛选模式。而目前发展成熟的高通量细胞筛选系统主要基于多孔板,存在细胞培养条件单一、耗时费力、试剂消耗量大等问题,且较难实现复杂的组合药物筛选。微流控技术作为一种在微米尺度通道中操纵和控制微流体的技术,具有微量、高效、高通量和自动化的优点,能较好地克服多孔板筛选系统的不足,为构建细胞高通量药物筛选系统提供了一种高效、可靠的技术手段。微流控系统在细胞培养材料、芯片结构设计和流体控制方面均可灵活变化,能更好地实现对细胞生长微环境的调控和模拟。文章综述了基于微流控技术的细胞水平高通量药物筛选系统的研究进展,按照不同的微流体操控模式,对基于灌注流、液滴和微阵列的3种类型的微流控细胞筛选系统进行了分类介绍,并分别总结了它们的优缺点,最后展望了微流控细胞水平高通量药物筛选系统的发展前景,提出了该领域目前存在的问题以及解决问题的方向。     

A method for DNA detection in a microchannel: fluid dynamics phenomena and optimization of microchannel structure

A simple DNA diagnosis method using microfluidics has been developed which requires simple and straightforward procedures such as injection of sample and probe DNA solutions. This method takes advantage of the highly accurate control of fluids in microchannels, and is superior to DNA microarray diagnosis methods due to its simplicity, highly quantitative determination, and high-sensitivity. The method is capable of detecting DNA hybridization for molecules as small as a 20 mer. This suggests the difference in microfluidic behavior between single strand DNA (ssDNA) and double stranded DNA (dsDNA). In this work, influence of both the inertial force exerted on DNA molecules and the diffusion of DNA molecules was investigated. Based on the determination of these parameters for both ssDNA and dsDNA by experiments, a numerical model describing the phenomena in the microchannel was designed. Computational simulation results using this model were in good agreement with previously reported experimental results. The simulation results showed that appropriate selection of the analysis point and the design of microchannel structure are important to bring out the diffusion and inertial force effects suitably and increase the sensitivity of the detection of DNA hybridization, that is, the analytical performance of the microfluidic DNA chip.     

MUC1 Aptamer-Capped Mesoporous Silica Nanoparticles for Navitoclax Resistance Overcoming in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. In the last years, navitoclax has emerged as a possible treatment for TNBC. Nevertheless, rapid navitoclax resistance onset has been observed thorough Mcl-1 overexpression. As a strategy to overcome Mcl-1-mediated resistance, herein we present a controlled drug co-delivery system based on mesoporous silica nanoparticles (MSNs) targeted to TNBC cells. The nanocarrier is loaded with navitoclax and the Mcl-1 inhibitor S63845 and capped with a MUC1-targeting aptamer ( apMUC1-MSNs(Nav/S63845) ). The apMUC1-capped nanoparticles effectively target TNBC cell lines and successfully induce apoptosis, overcoming navitoclax resistance. Moreover, navitoclax encapsulation protects platelets against apoptosis. These results point apMUC1-gated MSNs as suitable BH3 mimetics nanocarriers in the targeted treatment of MUC1-expressing TNBC.