Human Astrocyte Spheroids as Suitable In Vitro Screening Model to Evaluate Synthetic Cannabinoid MAM2201-Induced Effects on CNS.

There is growing concern about the consumption of synthetic cannabinoids (SCs), one of the largest groups of new psychoactive substances, its consequence on human health (general population and workers), and the continuous placing of new SCs on the market. Although drug-induced alterations in neuronal function remain an essential component for theories of drug addiction, accumulating evidence indicates the important role of activated astrocytes, whose essential and pleiotropic role in brain physiology and pathology is well recognized. The study aims to clarify the mechanisms of neurotoxicity induced by one of the most potent SCs, named MAM-2201 (a naphthoyl-indole derivative), by applying a novel three-dimensional (3D) cell culture model, mimicking the physiological and biochemical properties of brain tissues better than traditional two-dimensional in vitro systems. Specifically, human astrocyte spheroids, generated from the D384 astrocyte cell line, were treated with different MAM-2201 concentrations (1-30 µM) and exposure times (24-48 h). MAM-2201 affected, in a concentration- and time-dependent manner, the cell growth and viability, size and morphological structure, E-cadherin and extracellular matrix, CB1-receptors, glial fibrillary acidic protein, and caspase-3/7 activity. The findings demonstrate MAM-2201-induced cytotoxicity to astrocyte spheroids, and support the use of this human 3D cell-based model as species-specific in vitro tool suitable for the evaluation of neurotoxicity induced by other SCs.

Fluorescence lifetime imaging of intracellular magnesium content in live cells.

The first detailed analysis of FLIM applications for Mg cell imaging is presented. We employed the Mg-sensitive fluorescent dye named DCHQ5, a derivative of diaza-18-crown-6 ethers appended with two 8-hydroxyquinoline groups, to perform fluorescence lifetime imaging in control and Mg deprived SaOS-2 live cells, which contain different concentrations of magnesium. We found that the lifetime maps are almost uniform all over the cells and, most relevantly, we showed that the ratio of the amplitude terms is related to the magnesium intracellular concentration.

Single cell versus large population analysis: cell variability in elemental intracellular concentration and distribution.

The quantification of elemental concentration in cells is usually performed by analytical assays on large populations missing peculiar but important rare cells. The present article aims at comparing the elemental quantification in single cells and cell population in three different cell types using a new approach for single cells elemental analysis performed at sub-micrometer scale combining X-ray fluorescence microscopy and atomic force microscopy. The attention is focused on the light element Mg, exploiting the opportunity to compare the single cell quantification to the cell population analysis carried out by a highly Mg-selective fluorescent chemosensor. The results show that the single cell analysis reveals the same Mg differences found in large population of the different cell strains studied. However, in one of the cell strains, single cell analysis reveals two cells with an exceptionally high intracellular Mg content compared with the other cells of the same strain. The single cell analysis allows mapping Mg and other light elements in whole cells at sub-micrometer scale. A detailed intensity correlation analysis on the two cells with the highest Mg content reveals that Mg subcellular localization correlates with oxygen in a different fashion with respect the other sister cells of the same strain. Graphical abstract Single cells or large population analysis this is the question!

Magnesium Deprivation Potentiates Human Mesenchymal Stem Cell Transcriptional Remodeling.

Magnesium plays a pivotal role in energy metabolism and in the control of cell growth. While magnesium deprivation clearly shapes the behavior of normal and neoplastic cells, little is known on the role of this element in cell differentiation. Here we show that magnesium deficiency increases the transcription of multipotency markers and tissue-specific transcription factors in human adipose-derived mesenchymal stem cells exposed to a mixture of natural molecules, i.e., hyaluronic, butyric and retinoid acids, which tunes differentiation. We also demonstrate that magnesium deficiency accelerates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. We argue that magnesium deprivation generates a stressful condition that modulates stem cell plasticity and differentiation potential. These studies indicate that it is possible to remodel transcription in mesenchymal stem cells by lowering extracellular magnesium without the need for genetic manipulation, thus offering new hints for regenerative medicine applications.

Where is it and how much? Mapping and quantifying elements in single cells.

The biological function of a chemical element in cells not only requires the determination of its intracellular quantity, but also the spatial distribution of its concentration. Different strategies can be employed to quantify and map the intracellular concentration of elements in single cells. The assessment of the intracellular elemental concentration, which is the relevant information, requires the measurement of cell volume. This challenging and demanding task requires combining different techniques allowing gathering of both morphological and compositional information on the same cell. Moreover, the need to analyse samples more similar to their natural state requires complex hardware equipment, and supplementary efforts in preparation protocols. Nevertheless, the response to the question: "where is it and how much?" is worth all these efforts. This review aims at providing an insight into the recent and most advanced techniques and strategies for quantifying and mapping chemical elements in single cells. We describe and discuss indirect detection techniques (label based) which make use of fluorescent dyes, and direct ones (label free), such as particle induced X-ray emission, proton backscattering spectrometry, scanning transmission ion spectrometry, nano-secondary ion mass spectrometry, X-ray fluorescence microscopy, complemented by X-ray imaging.

Quantitative chemical imaging of the intracellular spatial distribution of fundamental elements and light metals in single cells.

We report a method that allows a complete quantitative characterization of whole single cells, assessing the total amount of carbon, nitrogen, oxygen, sodium, and magnesium and providing submicrometer maps of element molar concentration, cell density, mass, and volume. This approach allows quantifying elements down to 10(6) atoms/µm(3). This result was obtained by applying a multimodal fusion approach that combines synchrotron radiation microscopy techniques with off-line atomic force microscopy. The method proposed permits us to find the element concentration in addition to the mass fraction and provides a deeper and more complete knowledge of cell composition. We performed measurements on LoVo human colon cancer cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin. The comparison of LoVo-S and LoVo-R revealed different patterns in the maps of Mg concentration with higher values within the nucleus in LoVo-R and in the perinuclear region in LoVo-S cells. This feature was not so evident for the other elements, suggesting that Mg compartmentalization could be a significant trait of the drug-resistant cells.

A novel fluorescent chemosensor allows the assessment of intracellular total magnesium in small samples.

The present study investigated the analytical capabilities of a new fluorescent chemosensor, named DCHQ5, a phenyl derivative belonging to the family of diaza-crown-hydroxyquinolines, for the quantitative assessment of total intracellular Mg content. The results obtained were compared to the analytical performances of DCHQ1 - the parent probe of the series which so far was the only suitable species for the quantitative assessment of the intracellular total magnesium and showed comparable results to atomic absorption spectroscopy. Different protocols were tested in several cell lines both by flow cytometry and by steady state fluorescence spectroscopy assays. The results obtained support the possibility to use DCHQ5 to accurately quantify the intracellular total Mg in much smaller samples than DCHQ1, also displaying an increased stable intracellular staining. These features, combined with the high fluorescence enhancement upon cation binding, and the possibility to be excited both in the UV and visible region, make DCHQ5 a valuable and versatile analytical tool for Mg assessment in biological samples.

Adipose Stromal Cell Spheroids for Cartilage Repair: A Promising Tool for Unveiling the Critical Maturation Point.

Articular cartilage lacks intrinsic regenerative capabilities, and the current treatments fail to regenerate damaged tissue and lead only to temporary pain relief. These limitations have prompted the development of tissue engineering approaches, including 3D culture systems. Thanks to their regenerative properties and capacity to recapitulate embryonic processes, spheroids obtained from mesenchymal stromal cells are increasingly studied as building blocks to obtain functional tissues. The aim of this study was to investigate the capacity of adipose stromal cells to assemble in spheroids and differentiate toward chondrogenic lineage from the perspective of cartilage repair. Spheroids were generated by two different methods (3D chips vs. Ultra-Low Attachment plates), differentiated towards chondrogenic lineage, and their properties were investigated using molecular biology analyses, biophysical measurement of mass density, weight, and size of spheroids, and confocal imaging. Overall, spheroids showed the ability to differentiate by expressing specific cartilaginous markers that correlate with their mass density, defining a critical point at which they start to mature. Considering the spheroid generation method, this pilot study suggested that spheroids obtained with chips are a promising tool for the generation of cartilage organoids that could be used for preclinical/clinical approaches, including personalized therapy.

Manuka honey in combination with 5-Fluorouracil decreases physical parameters of colonspheres enriched with cancer stem-like cells and reduces their resistance to apoptosis.

The aim of the present work was to evaluate the in vitro effect of Manuka honey and its combination with 5-Fu, the most common drug used in the treatment of colon cancer, on the morphological and physical parameters of colonspheres enriched with cancer stem-like cells deriving from HCT-116 colon adenocarcinoma cell line and on the apoptosis rate. Manuka honey, alone and more in combination with 5-Fu, reduced the weight, the diameter and mass density of the spheroids and induced apoptosis through the downregulation of many apoptosis inhibitors, including IAPs (Livin, Survivin, XIAP), IGFs (IGF-I, IGF-II and IGF-IR) and HSPs (HSP-27, HSP-60 and HSP-70). These results led to a reduction in the survival ability of cancer stem-like cells, as well as to a chemosensitizing effect of honey towards 5-Fu, considering that apoptosis resistance is one of the main causes of cancer stem-like cells chemoresistance.

A new method for the study of biophysical and morphological parameters in 3D cell cultures: Evaluation in LoVo spheroids treated with crizotinib.

Three-dimensional (3D) culture systems like tumor spheroids represent useful in vitro models for drug screening and more broadly for cancer biology research, but the generation of uniform populations of spheroids remains challenging. The possibility to properly characterize spheroid properties would increase the reliability of these models. To address this issue different analysis were combined: i) a new device and relative analytical method for the accurate, simultaneous, and rapid measurement of mass density, weight, and size of spheroids, ii) confocal imaging, and iii) protein quantification, in a clinically relevant 3D model. The LoVo colon cancer cell line forming spheroids, treated with crizotinib (CZB) an ATP-competitive small-molecule inhibitor of the receptor tyrosine kinases, was employed to study and assess the correlation between biophysical and morphological parameters in both live and fixed cells. The new fluidic-based measurements allowed a robust phenotypical characterization of the spheroids structure, offering insights on the spheroids bulk and an accurate measurement of the tumor density. This analysis helps overcome the technical limits of the imaging that hardly penetrates the thickness of 3D structures. Accordingly, we were able to document that CZB treatment has an impact on mass density, which represents a key marker characterizing cancer cell treatment. Spheroid culture is the ultimate technology in drug discovery and the adoption of such precise measurement of the tumor characteristics can represent a key step forward for the accurate testing of treatment's potential in 3D in vitro models.

Microfluidic Tools for Enhanced Characterization of Therapeutic Stem Cells and Prediction of Their Potential Antimicrobial Secretome.

Antibiotic resistance is creating enormous attention on the development of new antibiotic-free therapy strategies for bacterial diseases. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials and included in several cell-therapy protocols. Together with the well-known immunomodulatory and regenerative potential of the MSC secretome, these cells have shown direct and indirect anti-bacterial effects. However, the low reproducibility and standardization of MSCs from different sources are the current limitations prior to the purification of cell-free secreted antimicrobial peptides and exosomes. In order to improve MSC characterization, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. We discuss the potential of emerging microfluidic technologies providing new insights into density, shape, and size of live cells, starting from heterogeneous or 3D cultured samples. The prospective application of these technologies to studying MSC populations may contribute to developing new biopharmaceutical strategies with a view to naturally overcoming bacterial defense mechanisms.

Physical Characterization of Colorectal Cancer Spheroids and Evaluation of NK Cell Infiltration Through a Flow-Based Analysis.

To improve pathogenetic studies in cancer development and reliable preclinical testing of anti-cancer treatments, three-dimensional (3D) cultures, including spheroids, have been widely recognized as more physiologically relevant in vitro models of in vivo tumor behavior. Currently, the generation of uniformly sized spheroids is still challenging: different 3D cell culture methods produce heterogeneous populations in dimensions and morphology, that may strongly influence readouts reliability correlated to tumor growth rate or antitumor natural killer (NK) cell-mediated cytotoxicity. In this context, an increasing consensus claims the integration of microfluidic technologies within 3D cell culture, as the physical characterization of tumor spheroids is unavoidably demanded to standardize protocols and assays for in vitro testing. In this paper, we employed a flow-based method specifically conceived to measure weight, size and focused onto mass density values of tumor spheroids. These measurements are combined with confocal and digital imaging of such samples. We tested the spheroids of four colorectal cancer (CRC) cell lines that exhibit statistically relevant differences in their physical characteristics, even though starting from the same cell seeding density. These variations are seemingly cell line-dependent and associated with the number of growing cells and the degree of spheroid compaction as well, supported by different adenosine-triphosphate contents. We also showed that this technology can estimate the NK cell killing efficacy by measuring the weight loss and diameter shrinkage of tumor spheroids, alongside with the commonly used cell viability in vitro test. As the activity of NK cells relies on their infiltration rate, the in vitro sensitivity of CRC spheroids proved to be exposure time- and cell line-dependent with direct correlation to the cell viability reduction. All these functional aspects can be measured by the system and are documented by digital image analysis. In conclusion, this flow-based method potentially paves the way towards standardization of 3D cell cultures and its early adoption in cancer research to test antitumor immune response and set up new immunotherapy strategies.

A Reliable Flow-Based Method for the Accurate Measure of Mass Density, Size and Weight of Live 3D Tumor Spheroids.

Gathering precise information on mass density, size and weight of cells or cell aggregates, is crucial for applications in many biomedical fields with a specific focus on cancer research. Although few technical solutions have been presented for single-cell analysis, literature does not cover this aspect for 3D models such as spheroids. Since the research interest on such samples is notably rising, here we describe a flow-apparatus, and the associated physical method and operative protocol for the accurate measurements of mass density, size and weight. The technique is based on the detection of the terminal velocity of a free-falling sample into a specifically conceived analysis flow-channel. Moreover, in order to demonstrate the accuracy and precision of the presented flow-device, analyses were initially carried out on standardized polystyrene beads. Finally, to display the application of the proposed system for biological samples, mass density, size and weight of live SW620 tumor spheroids were analyzed. The combined measurements of such parameters can represent a step toward a deeper understanding of 3D culture models.

Correction to Chemical Fingerprint of Zn-Hydroxyapatite in the Early Stages of Osteogenic Differentiation.

[This corrects the article DOI: 10.1021/acscentsci.9b00509.].

Chemical Fingerprint of Zn-Hydroxyapatite in the Early Stages of Osteogenic Differentiation.

The core knowledge about biomineralization is provided by studies on the advanced phases of the process mainly occurring in the extracellular matrix. Here, we investigate the early stages of biomineralization by evaluating the chemical fingerprint of the initial mineral nuclei deposition in the intracellular milieu and their evolution toward hexagonal hydroxyapatite. The study is conducted on human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days, exploiting laboratory X-ray diffraction techniques and cutting-edge developments of synchrotron-based 2D and 3D cryo-X-ray microscopy. We demonstrate that biomineralization starts with Zn-hydroxyapatite nucleation within the cell, rapidly evolving toward hexagonal hydroxyapatite crystals, very similar in composition and structure to the one present in human bone. These results provide experimental evidence of the germinal role of Zn in hydroxyapatite nucleation and foster further studies on the intracellular molecular mechanisms governing the initial phases of bone tissue formation.

Overexpression of the mitochondrial Mg channel MRS2 increases total cellular Mg concentration and influences sensitivity to apoptosis.

The mechanism of action of the mitochondrial Mg channel MRS2 and its involvement in cell viability remain unclear. Deletion of MRS2 has been reported to abolish Mg influx into mitochondria, to induce functional defects in mitochondrial organelles, and to result in cell death. We evaluated whether MRS2 expression had an impact on total Mg cellular content by inducing the overexpression of MRS2 in HEK-293 cells. We observed a remarkable increase of total intracellular Mg concentration in cells overexpressing MRS2 compared with control cells. In order to investigate whether and in what manner the detected Mg increment was involved in the MRS2 influence on cell viability, we treated MRS2-overexpressing cells with two known apoptotic inducers. We found that cells overexpressing the MRS2 channel became less responsive to these pharmacological insults. Our experimental evidence indicates that the MRS2 channel controls overall intracellular Mg levels, the alteration of which might have a role in the molecular signaling leading to apoptotic cell death.

Synthesis of a highly Mg2+-selective fluorescent probe and its application to quantifying and imaging total intracellular magnesium.

Magnesium plays a crucial role in many physiological functions and pathological states. Therefore, the evolution of specific and sensitive tools capable of detecting and quantifying this element in cells is a very desirable goal in biological and biomedical research. We developed a Mg2+-selective fluorescent dye that can be used to selectively detect and quantify the total magnesium pool in a number of cells that is two orders of magnitude smaller than that required by flame atomic absorption spectroscopy (F-AAS), the reference analytical method for the assessment of cellular total metal content. This protocol reports itemized steps for the synthesis of the fluorescent dye based on diaza-18-crown-6-hydroxyquinoline (DCHQ5). We also describe its application in the quantification of total intracellular magnesium in mammalian cells and the detection of this ion in vivo by confocal microscopy. The use of in vivo confocal microscopy enables the quantification of magnesium in different cellular compartments. As an example of the sensitivity of DCHQ5, we studied the involvement of Mg2+ in multidrug resistance in human colon adenocarcinoma cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin, and found that the concentration was higher in LoVo-R cells. The time frame for DCHQ5 synthesis is 1-2 d, whereas the use of this dye for total intracellular magnesium quantification takes 2.5 h and for ion bioimaging it takes 1-2 h.

The different expression of TRPM7 and MagT1 impacts on the proliferation of colon carcinoma cells sensitive or resistant to doxorubicin.

The processes leading to anticancer drug resistance are not completely unraveled. To get insights into the underlying mechanisms, we compared colon carcinoma cells sensitive to doxorubicin with their resistant counterpart. We found that resistant cells are growth retarded, and show staminal and ultrastructural features profoundly different from sensitive cells. The resistant phenotype is accompanied by the upregulation of the magnesium transporter MagT1 and the downregulation of the ion channel kinase TRPM7. We demonstrate that the different amounts of TRPM7 and MagT1 account for the different proliferation rate of sensitive and resistant colon carcinoma cells. It remains to be verified whether they are also involved in the control of other "staminal" traits.

Coumarin derivatives as potential antitumor agents: Growth inhibition, apoptosis induction and multidrug resistance reverting activity.

A small library of coumarins, carrying butynyl-amino chains, was synthesized continuing our studies in the field of MDR reverting ageEnts and in order to obtain multipotent agents to combat malignancies. In particular, the reported anticancer and chemopreventive natural product 7-isopentenyloxycoumarin was linked to different terminal amines, selected on the basis of our previously reported results. The anticancer behaviour and the MDR reverting ability of the new compounds were evaluated on human colon cancer cells, particularly prone to develop the MDR phenotype. Some of the new derivatives showed promising effects, directly acting as cytotoxic compounds and/or counteracting MDR phenomenon. Compound 1e emerged as the most interesting of this series, showing a multipotent biological profile and suggesting that conjugation of an appropriate coumarin core with a properly selected butynyl-amino chain allows to obtain novel hybrid molecules endowed with improved in vitro antitumor activity.

Effects of supplementation with different Mg salts in cells: is there a clue?

The differing bioavailability of magnesium salts remains an open question, both at the cellular and systemic level. However, this issue is relevant for identifying the most effective magnesium supplement. We compared the effects of three widely used magnesium salts: MgSO4, MgCl2 and Mg pidolate, on the proliferation of four human cell types: promyelocytic leukaemia HL60, osteoblast-like Saos-2 and U-2 OS, and endothelial cells from the umbilical vein. The three magnesium salts had no effect on endothelial and leukemic cell growth, but magnesium pidolate impaired cell growth in osteoblast-like cells. In particular, in Saos-2 cells, 1 mM pidolate induced a slight accumulation of cells in the G0/G1 phase of the cell cycle and, in parallel, an early rise in intracellular calcium and a late decrease in intracellular magnesium content. Interestingly, when cultured in 5 mM magnesium pidolate, Saos-2 cells grew as fast as the controls. Moreover, intracellular magnesium and calcium concentrations did not vary. These results suggest a lower bioavailability of magnesium pidolate in osteoblast-like cells.