Combinatorial Therapeutic Effect of Inhibitors of Aldehyde Dehydrogenase and Mitochondrial Complex I,and the Chemotherapeutic Drug,Temozolomide against Glioblastoma Tumorspheres

Resident cancer cells with stem cell-like features induce drug tolerance, facilitating survival of glioblastoma (GBM). We previously showed that strategies targeting tumor bioenergetics present a novel emerging avenue for treatment of GBM. The objective of this study was to enhance the therapeutic effects of dual inhibition of tumor bioenergetics by combination of gossypol, an aldehyde dehydrogenase inhibitor, and phenformin, a biguanide compound that depletes oxidative phosphorylation, with the chemotherapeutic drug, temozolomide (TMZ), to block proliferation, stemness, and invasiveness of GBM tumorspheres (TSs). Combination therapy with gossypol, phenformin, and TMZ induced a significant reduction in ATP levels, cell viability, stemness, and invasiveness compared to TMZ monotherapy and dual therapy with gossypol and phenformin. Analysis of differentially expressed genes revealed up-regulation of genes involved in programmed cell death, autophagy, and protein metabolism and down-regulation of those associated with cell metabolism, cycle, and adhesion. Combination of TMZ with dual inhibitors of tumor bioenergetics may, therefore, present an effective strategy against GBM by enhancing therapeutic effects through multiple mechanisms of action.     

On-chip anticancer drug test of regular tumor spheroids formed in microwells by a distributive microchannel network

This paper proposes a new cytotoxicity assay in a microfluidic device with microwells and a distributive microfluidic channel network for the formation of cancer cell spheroids. The assay can generate rapid and uniform cell clusters in microwells and test in situ cytotoxicity of anticancer drugs including sequential drug treatments, long term culture of spheroids and cell viability assays. Inlet ports are connected to the microwells by a hydraulic resistance network. This uniform distribution of cell suspensions results in regular spheroid dimensions. Injected cancer cells were trapped in microwells, and aggregated into tumor spheroids within 3 days. A cytotoxicity test of the spheroids in microwells was subsequently processed in the same device without the extraction of cells. The in situ cytotoxicity assay of tumor spheroids in microwells was comparable with the MTT assay on hanging drop spheroids using a conventional 96-well plate. It was observed that the inhibition rate of the spheroids was less than that in the 2D culture dish and the effect on tumor spheroids was different depending on the anticancer drug. This device could provide a convenient in situ assay tool to assess the cytotoxicity of anticancer drugs on tumor spheroids, offering more information than the conventional 2D culture plate.     

A Coculture Based, 3D Bioprinted Ovarian Tumor Model Combining Cancer Cells and Cancer Associated Fibroblasts

Ovarian cancer remains a major public health issue due to its poor prognosis. To develop more effective therapies, it is crucial to set-up reliable models that closely mimic the complexity of the ovarian tumor's microenvironment. 3D bioprinting is currently a promising approach to build heterogenous and reproducible cancer models with controlled shape and architecture. However, this technology is still poorly investigated to model ovarian tumors. In this study, a 3D bioprinted ovarian tumor model combining cancer cells (SKOV-3) and cancer associated fibroblasts (CAFs) are described. The resulting tumor models show their ability to maintain cell viability and proliferation. Cells are observed to self-assemble in heterotypic aggregates. Moreover, CAFs are observed to be recruited and to circle cancer cells reproducing an in vivo process taking place in the tumor microenvironment. Interestingly, this approach also shows its ability to rapidly generate a high number of reproducible tumor models that can be subjected to usual characterizations (cell viability and metabolic activity; histology and immunological studies; and real-time imaging). Therefore, these ovarian tumor models can be an interesting tool for high throughput drug screening applications.     

Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-β2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.     

Effects of electrode surface modification with chlorotoxin on patterning single glioma cells

A microchip patterned with arrays of single cancer cells can be an effective platform for the study of tumor biology, medical diagnostics, and drug screening. However, patterning and retaining viable single cancer cells on defined sites of the microarray can be challenging. In this study we used a tumor cell-specific peptide, chlorotoxin (CTX), to mediate glioma cell adhesion on arrays of gold microelectrodes and investigated the effects of three surface modification schemes for conjugation of CTX to the microelectrodes on single cell patterning, which include physical adsorption, covalent bonding mediated by N-hydroxysuccinimide (NHS), and covalent bonding via crosslinking succinimidyl iodoacetate and Traut's (SIA-Traut) reagents. The CTX immobilization to microelectrodes was confirmed by high-resolution X-ray photoelectron spectroscopy. Physically adsorbed CTX showed better support for cell adhesion and is more effective in confining adhered cells on the electrodes than covalently-bound CTX. Furthermore, cell adhesion and spreading on microelectrodes were quantified in real-time by impedance measurements, which revealed an impedance signal from physically adsorbed CTX electrodes four times greater than the signal from covalently-bound CTX electrodes.     

电场或电磁场和光动力的协同效应对癌细胞失活和坏死的作用(英文)

应用弱正弦波电磁场改变细胞膜的穿透性并引起其失活和坏死.研究中,将人类癌细胞U 937和K 562放置于强度为10mT和39mT(50Hz)的正弦波电磁场内,并依次结合细胞毒素放线菌素 C以及其独特的光动力活性分别进行试验.     

Studies of anticancer drug cytotoxicity based on long‐term HepG2 spheroid culture in a microfluidic system

Cell‐on‐a‐chip systems have become promising devices to study the effectiveness of new anticancer drugs recently. Several microdevices for liver cancer culture and evaluation of the drug cytotoxicity have been reported. However, there are still no proven reports about high‐throughput and simple methods for the evaluation of drug cytotoxicity on liver cancer cells. The paper presents the results of the effects of the anticancer drug (5‐fluorouracil, 5‐FU) on the HepG2 spheroids as a model of liver cancer. The experiments were based on the long‐term 3D spheroid culture in the microfluidic system and monitoring of the effect of 5‐FU at two selected concentrations (0.5 mM and 1.0 mM). Our investigations have shown that the initial size of the spheroids has influence on the drug effect. With the increase of the spheroids diameter, the drug resistance (for the two tested 5‐FU concentrations) decreases. This phenomenon was observed both through cells metabolism analysis, as well as changes in spheroids sizes. In our research, we have shown that the lower 5‐FU (0.5 mM) concentration causes higher decrease in HepG2 spheroids viability. Moreover, due to the microsystem construction, we observe the drug resistance effect (10th day of culture) regardless of the initial size of the created spheroids and the drug concentration.     

Mitochondria Targeted Nanoparticles Potentiate Tumor Chemo-Phototherapy by Toxic Oxidative Stress Mediated Oxeiptosis

Insufficient accumulation of drug at the tumor site and the low drug response are the main reason for the unsatisfactory effect of cancer therapy. Delivery drugs exquisitely to subcellular level can be employed to reduce side effects, and expand the therapeutic window. Herein, a triphenylphosphine (TPP) modified lipid nanoparticles is designed which are loaded with the photosensitizer indocyanine green (ICG) and chemotherapeutic paclitaxel (PTX) for mitochondria-targeted chemo-phototherapy. Owing to the movement of majority mitochondria along microtubules in cytoplasm, mitochondrial targeting may enable PTX to act more effectively. Meanwhile, the existence of chemo-drug potentiates the phototherapy to achieve synergistic anti-tumor activity. As expected, mitochondria targeting nanomedicine (M-ICG-PTX NPs) showed improved mitochondria targeted cellular distribution and enhanced cell cytotoxicity in vitro. Also, M-ICG-PTX NPs exhibited higher tumor growth inhibition ability by promoting cell apoptosis and oxeiptosis pathway, and high effective inhibition of primary tumor growth and tumor metastasis. Taken together, M-ICG-PTX NPs may be promising nanoplatforms to achieve potent therapeutic effect for the combination of chemo- and photo-therapy (PTT).     

Tubule Nanoclay‐Organic Heterostructures for Biomedical Applications

Natural halloysite nanotubes (HNTs) show unique hollow structure, high aspect ratio and adsorption ability, good biocompatibility, and low toxicity, which allow for various biomedical applications in the diagnosis and treatment of diseases. Here, advances in self‐assembly of halloysite for cell capturing and bacterial proliferation, coating on biological surfaces and related drug delivery, bone regeneration, bioscaffolds, and cell labeling are summarized. The in vivo toxicity of these clay nanotubes is discussed. Halloysite allows for 10–20% drug loading and can extend the delivery time to 10–100 h. These drug‐loaded nanotubes are doped into the polymer scaffolds to release the loaded drugs. The rough surfaces fabricated by self‐assembly of the clay nanotubes enhance the interactions with tumor cells, and the cell capture efficacy is significantly improved. Since halloysite has no toxicity toward microorganisms, the bacteria composed within these nanotubes can be explored in oil/water emulsion for petroleum spilling bioremediation. Coating of living cells with halloysite can control the cell growth and is not harmful to their viability. Quantum dots immobilized on halloysite were employed for cell labeling and imaging. The concluding academic results combined with the abundant availability of these natural nanotubes promise halloysite applications in personal healthcare and environmental remediation.     

Determination of DNA Hypermethylation Using Anti‐cancer Drug‐Temozolomide

An electrochemical drug‐DNA biosensor was developed for the detection of interaction between the anti‐cancer drug, Temozolomide (TMZ), and DNA sequences by using Differential Pulse Voltammetry at the graphite electrode surfaces. TMZ is a pro‐drug and an alkylating agent that crosses the blood‐brain barrier, so it is mainly used for brain cancers treatment. In this study, we aim to develop a‐proof‐of‐concept study to investigate the effect of TMZ on formerly methylated DNA sequences since TMZ shows its anti‐cancer activity by methylating the DNA. Interaction between TMZ and DNA causes localized distortion of DNA away from an idealized B‐form, resulting in a wider major groove and greater steric accessibility of functional groups in the base of the groove. According to the results, TMZ behaves as a ‘hybridization indicator’ because of its different electrochemical behavior to different strands of DNA. After interaction with TMZ, hybrid (double stranded DNA‐dsDNA) signals decreased dramatically whereas probe (single stranded DNA‐ssDNA) and control signals remain almost unchanged. The signal differences enabled us to distinguish ssDNA and dsDNA without using a label or tag. It is the first study to demonstrate the interaction between the TMZ and dsDNA created from probe and target. We use specific oligonucleotides sequences instead of using long dsDNA sequences.     

Cellular impedance biosensors for drug screening and toxin detection

Cell-based impedance biosensing is an emerging technology that can be used to non-invasively and instantaneously detect and analyze cell responses to chemical and biological agents. This article highlights the fabrication and measurement technologies of cell impedance sensors, and their application in toxin detection and anti-cancer drug screening. We start with an introduction that describes the capability and advantages of cell-based sensors over conventional sensing technology, followed by a discussion of the influence of cell adhesion, spreading and viability during cell patterning on the subsequent impedance measurements and sensing applications. We then present an electronic circuit that models the cell-electrode system, by which the cellular changes can be detected in terms of impedance changes of the circuit. Finally, we discuss the current status on using cell impedance sensors for toxin detection and anti-cancer drug screening.     

Co-culture of tumor spheroids and monocytes in a collagen matrix-embedded microfluidic device to study the migration of breast cancer cells

A 3D co-culture microfluidic device was developed to study the effects of ECM stiffness and TAMs on tumor cells migration.     

Short peptides enhance single cell adhesion and viability on microarrays

Single cell patterning holds important implications for biology, biochemistry, biotechnology, medicine, and bioinformatics. The challenge for single cell patterning is to produce small islands hosting only single cells and retaining their viability for a prolonged period of time. This study demonstrated a surface engineering approach that uses a covalently bound short peptide as a mediator to pattern cells with improved single cell adhesion and prolonged cellular viability on gold patterned SiO2 substrates. The underlying hypothesis is that cell adhesion is regulated by the type, availability, and stability of effective cell adhesion peptides, and thus covalently bound short peptides would promote cell spreading and, thus, single cell adhesion and viability. The effectiveness of this approach and the underlying mechanism for the increased probability of single cell adhesion and prolonged cell viability by short peptides were studied by comparing cellular behavior of human umbilical cord vein endothelial cells on three model surfaces whose gold electrodes were immobilized with fibronectin, physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently bound Lys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and binding properties were characterized by reflectance Fourier transform infrared spectroscopy. Both short peptides were superior to fibronectin in producing adhesion of only single cells, whereas the covalently bound peptide also reduced apoptosis and necrosis of adhered cells. Controlling cell spreading by peptide binding domains to regulate apoptosis and viability represents a fundamental mechanism in cell-materials interaction and provides an effective strategy in engineering arrays of single cells.     

Enhanced stability and activity of temozolomide in primary glioblastoma multiforme cells with cucurbit[n]uril

Temozolomide (TMZ) is the primary chemotherapeutic agent for treatment of glioblastoma multiforme (GBM) yet it has a fast rate of degradation under physiological conditions to the 'active' MTIC, which has poor penetration of the blood-brain barrier and cellular absorption. Herein we have demonstrated binding of TMZ within the cavity of nano-container cucurbit[7]uril, resulting in a decreased rate of drug degradation. Prolonging the lifetime of the TMZ under physiological conditions through encapsulation dramatically improved the drug's activity against primary GBM cell lines as more TMZ could be absorbed by the cells before degradation. This work can potentially lead to increases in the drug's propensity for crossing the blood-brain barrier and absorption into the GBM cells, thereby increasing the efficacy of this chemotherapy.     

Delivery of ROS Generating Anthraquinones Using Reduction‐Responsive Peptide‐Based Nanoparticles

In order to limit the side effects associated with antitumor drugs such as doxorubicin, nanosized drug‐delivery systems capable of selectively delivering and releasing the drug in the diseased tissue are required. We describe nanoparticles (NPs), self‐assembled from a reduction responsive amphiphilic peptide, capable of entrapping high amounts of a redox active anticancer drug candidate and releasing it in presence of a reducing agent. This system shows a high entrapment efficiency with up to 15 mg drug per gram of peptide (5.8 mol‐%). Treatment of the NPs with reducing agent results in the disassembly of the NPs and release of the drug molecules. A reduction in cell viability is observed at drug concentrations above 250 nm in HEK293T and HeLa cell lines. This drug delivery system has potential for targeting tumor sites via the EPR effect while taking advantage of the increased reduction potential in tumor microenvironment.     

Use of cellular electrical impedance sensing to assess in vitro cytotoxicity of anticancer drugs in a human kidney cell nephrotoxicity model

Nephrotoxicity is one of the major concerns for anticancer drug safety because most drugs are metabolized and excreted by the kidneys. Convenient tools able to perform rapid in vitro cytotoxicity analysis and identify drug side effects in kidney cells during early phases of drug discovery could be beneficial to drug development programs. Here we developed an electrical cell-substrate impedance sensing system (ECIS) capable of continuously measuring the dosage and time response of human proximal tubular epithelial (HK2) cells exposed to four drugs throughout the experimental period. These drugs induced HK2 cell apoptosis/death in a dose-dependent manner, although with very different dose-response effects. DDP (50 μM) was the most cytotoxic and induced obvious HK2 cell apoptosis rapidly after exposure. The other three drugs had much lower cytotoxicity, even at concentrations approaching 1 mM. The results obtained from our ECIS system correlated well with conventional in vitro assays such as flow cytometry and cell viability assays. Notably, the continuous and automatic measurements provided by ECIS system allow for better resolution for drugs with different temporal toxicity profiles. Furthermore, we investigated the effect of DDP's antidotes, glutathione and sodium subsulfite, on DDP-induced cytotoxicity, both of which decreased nephrotoxicity of DDP in a dose-dependent manner. Overall this study illustrates the convenience of ECIS for direct, continuous assessment of the cytotoxicity of anticancer drugs in vitro. ECIS has the potential to become a useful, non-invasive analytical method for early evaluation of drugs and antidotes of toxins.     

An on-chip intestine-liver model for multiple drugs absorption and metabolism behavior simulation

A double-layer microfluidic chip integrated with a hollow fiber(HF)was developed to reconstitute the intestine-liver functionality for studying the absorption and metabolism of combination drugs.Caco-2 cells were inoculated in the HF cavity at the top of the serpentine channel to simulate the intestinal tissue for drug absorption and transport studied,and Hep G2 cells,seeded in the bottom chamber,were used to mimic the liver for metabolism-related studies.Genistein and dacarbazine were selected for combination drug therapy and its effects on cell viability,hepatotoxicity,and cell cycle arrest under drug-conditioned culture were investigated.The results suggested that the combined concentration below-100μg/m L had no significant inhibitory effect on Hep G2 cell viability,and therefore Hep G2 cells maintained their drug metabolism ability.When the drug concentration was increased above 250μg/m L,Hep G2 cells underwent apoptosis.Detection of metabolites by mass spectrometry proved the effective metabolism in the microchip model.This dynamic,co-culture microchip successfully provided a podium for long-term observation of absorption,transport,and metabolism of combination drugs,and could be an effective in vitro simulation model for further clinical research.     

Control of Three‐Dimensional Cell Adhesion by the Chirality of Nanofibers in Hydrogels

In the three‐dimensional (3D) extracellular matrix (ECM), the influence of nanofiber chirality on cell behavior is very important; the helical nanofibrous structure is closely related to the relevant biological events. Herein, we describe the use of the two enantiomers of a 1,4‐benzenedicarboxamide phenylalanine derivative as supramolecular gelators to investigate the influence of the chirality of nanofibers on cell adhesion and proliferation in three dimensions. It was found that left‐handed helical nanofibers can increase cell adhesion and proliferation, whereas right‐handed nanofibers have the opposite effect. These effects are ascribed to the mediation of the stereospecific interaction between chiral nanofibers and fibronectin. The results stress the crucial role of the chirality of nanofibers on cell‐adhesion and cell‐proliferation behavior in 3D environments.     

Self‐Folded Hydrogel Tubes for Implantable Muscular Tissue Scaffolds

Programming materials with tunable physical and chemical interactions among its components pave the way of generating 3D functional active microsystems with various potential applications in tissue engineering, drug delivery, and soft robotics. Here, the development of a recapitulated fascicle‐like implantable muscle construct by programmed self‐folding of poly(ethylene glycol) diacrylate hydrogels is reported. The system comprises two stacked layers, each with differential swelling degrees, stiffnesses, and thicknesses in 2D, which folds into a 3D tube together. Inside the tubes, muscle cell adhesion and their spatial alignment are controlled. Both skeletal and cardiac muscle cells also exhibit high viability, and cardiac myocytes preserve their contractile function over the course of 7 d. Integration of biological cells with smart, shape‐changing materials could give rise to the development of new cellular constructs for hierarchical tissue assembly, drug testing platforms, and biohybrid actuators that can perform sophisticated tasks.     

In Situ Scatheless Cell Detachment Reveals Correlation between Adhesion Strength and Viability at Single‐Cell Resolution

Single‐cell biology provides insights into some of the most fundamental processes in biology and promotes the understanding of life's mysteries. As the technologies to study single‐cells expand, they will require sophisticated analytical tools to make sense of various behaviors and components of single‐cells as well as their relations in the adherent tissue culture. In this paper, we revealed cell heterogeneity and uncovered the connections between cell adhesion strength and cell viability at single‐cell resolution by extracting single adherent cells of interest from a standard tissue culture by using a microfluidic chip‐based live single‐cell extractor (LSCE). We believe that this method will provide a valuable new tool for single‐cell biology.