Multifaceted interactions between cancer cells and glial cells in brain metastasis.

Cancer brain metastasis has a poor prognosis, is commonly observed in clinical practice, and the number of cases is increasing as overall cancer survival improves. However, experiments in mouse models have shown that brain metastasis itself is an inefficient process. One reason for this inefficiency is the brain microenvironment, which differs significantly from that of other organs, making it difficult for cancer cells to adapt. The brain microenvironment consists of unique resident cell types such as neurons, oligodendrocytes, astrocytes, and microglia. Accumulating evidence over the past decades suggests that the interactions between cancer cells and glial cells can positively or negatively influence the development of brain metastasis. Nevertheless, elucidating the complex interactions between cancer cells and glial cells remains challenging, in part due to the limitations of existing experimental models for glial cell culture. In this review, we first provide an overview of glial cell culture methods and then examine recent discoveries regarding the interactions between brain metastatic cancer cells and the surrounding glial cells, with a special focus on astrocytes and microglia. Finally, we discuss future perspectives for understanding the multifaceted interactions between cancer cells and glial cells for the treatment of metastatic brain tumors.

Profile of miRNAs in small extracellular vesicles released from glioblastoma cells treated by boron neutron capture therapy.

PURPOSE: Boron neutron capture therapy (BNCT) is a tumor cell-selective particle-radiation therapy. In BNCT, administered p-boronophenylalanine (BPA) is selectively taken up by tumor cells, and the tumor is irradiated with thermal neutrons. High-LET α-particles and recoil 7Li, which have a path length of 5-9 µm, are generated by the capture reaction between 10B and thermal neutrons and selectively kill tumor cells that have uptaken 10B. Although BNCT has prolonged the survival time of malignant glioma patients, recurrences are still to be resolved. miRNAs, that are encapsulated in small extracellular vesicles (sEVs) in body fluids and exist stably may serve critical role in recurrence. In this study, we comprehensively investigated microRNAs (miRNAs) in sEVs released from post-BNCT glioblastoma cells. METHOD: Glioblastoma U87 MG cells were treated with 25 ppm of BPA in the culture media and irradiated with thermal neutrons. After irradiation, they were plated into dishes and cultured for 3 days in the 5% CO2 incubator. Then, sEVs released into the medium were collected by column chromatography, and miRNAs in sEVs were comprehensively investigated using microarrays. RESULT: An increase in 20 individual miRNAs (ratio > 2) and a decrease in 2 individual miRNAs (ratio < 0.5) were detected in BNCT cells compared with non-irradiated cells. Among detected miRNAs, 20 miRNAs were associated with worse prognosis of glioma in Kaplan Meier Survival Analysis of overall survival in TCGA. CONCLUSION: These miRNA after BNCT may proceed tumors, modulate radiation resistance, or inhibit invasion and affect the prognosis of glioma.

MYEOV overexpression induced by demethylation of its promoter contributes to pancreatic cancer progression via activation of the folate cycle/c-Myc/mTORC1 pathway.

BACKGROUND: While molecular targeted drugs and other therapies are being developed for many tumors, pancreatic cancer is still considered to be the malignant tumor with the worst prognosis. We started this study to identify prognostic genes and therapeutic targets of pancreatic cancer. METHODS: To comprehensively identify prognostic genes in pancreatic cancer, we investigated the correlation between gene expression and cancer-specific prognosis using transcriptome and clinical information datasets from The Cancer Genome Atlas (TCGA). In addition, we examined the effects of the suppression of candidate prognostic genes in pancreatic cancer cell lines. RESULT: We found that patients with high expression levels of MYEOV, a primate-specific gene with unknown function, had significantly shorter disease-specific survival times than those with low expression levels. Cox proportional hazards analysis revealed that high expression of MYEOV was significantly associated with poor survival and was an independent prognostic factor for disease-specific survival in pancreatic cancer patients. Analysis of multiple cancer samples revealed that the MYEOV promoter region is methylated in noncancer tissues but is demethylated in tumors, causing MYEOV overexpression in tumors. Notably, the knockdown of MYEOV suppressed the expression of MTHFD2 and other folate metabolism-related enzyme genes required for the synthesis of amino acids and nucleic acids and also restored the expression of c-Myc and mTORC1 repressors. CONCLUSION: There is a significant correlation between elevated MYEOV expression and poor disease-specific survival in pancreatic cancer patients. MYEOV enhances the activation of several oncogenic pathways, resulting in the induction of pancreatic cancer cell proliferation. Overall, MYEOV acts as an oncogene in pancreatic cancer. Furthermore, MYEOV may be a prognostic biomarker and serve as an 'actionable' therapeutic target for pancreatic cancers.

ERK Activity Imaging During Migration of Living Cells In Vitro and In Vivo.

Extracellular signal-regulated kinase (ERK) is a major downstream factor of the EGFR-RAS-RAF signalling pathway, and thus the role of ERK in cell growth has been widely examined. The development of biosensors based on fluorescent proteins has enabled us to measure ERK activities in living cells, both after growth factor stimulation and in its absence. Long-term imaging unexpectedly revealed the oscillative activation of ERK in an epithelial sheet or a cyst in vitro. Studies using transgenic mice expressing the ERK biosensor have revealed inhomogeneous ERK activities among various cell species. In vivo Förster (or fluorescence) resonance energy transfer (FRET) imaging shed light on a novel role of ERK in cell migration. Neutrophils and epithelial cells in various organs such as intestine, skin, lung and bladder showed spatio-temporally different cell dynamics and ERK activities. Experiments using inhibitors confirmed that ERK activities are required for various pathological responses, including epithelial repair after injuries, inflammation, and niche formation of cancer metastasis. In conclusion, biosensors for ERK will be powerful and valuable tools to investigate the roles of ERK in situ.

Active K-RAS induces the coherent rotation of epithelial cells: A model for collective cell invasion in vitro.

At the invasive front of adenocarcinomas, single cells and multicellular structures exist; the latter include glands and cell clusters, such as tumor buddings and poorly differentiated clusters. Recent reports suggest the importance of collective cell migration in metastasis; however, it is technically difficult to observe the movement of multicellular structures in vivo. We utilized MDCK cells as a model for epithelial cells and established a method to quantify their motility in 3D structures in vitro. A single MDCK cell grows as a cell cluster in the gel and later proliferates and forms a cyst. Active K-RAS expression induced rotation of both the cell clusters and the cysts. The rotation speed of cell clusters was 4 times higher than that of cysts. The screening of inhibitors for their effects on cell clusters and cysts revealed that cyclin B1 and ß-catenin were the key molecules for their rotation, respectively. Regulators for cyst rotation, such as vorinostat and ß-catenin, were not effective for inducing cell cluster rotation. These results indicate that the signaling pathways of cell dynamics are different between cell clusters and cysts. As cell clusters are related to lymph node involvement and the prognosis of various carcinomas, our in vitro quantitative system may be useful for the screening of drugs to prevent lymphatic invasion.

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy.

Förster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena.

Fluctuation of Rac1 activity is associated with the phenotypic and transcriptional heterogeneity of glioma cells.

Phenotypic heterogeneity of cancer cells is caused not only by genetic and epigenetic alterations but also by stochastic variation of intracellular signaling molecules. Using cells that stably express Förster resonance energy transfer (FRET) biosensors, we show here a correlation between a temporal fluctuation in the activity of Rac1 and the invasive properties of C6 glioma cells. By using long-term time-lapse imaging, we found that Rac1 activity in C6 glioma cells fluctuated over a timescale that was substantially longer than that of the replication cycle. Because the relative level of Rac1 activity in each cell was unaffected by a suspension-adhesion procedure, we were able to sort C6 glioma cells according to the levels of Rac1 activity, yielding Rac1(high) and Rac1(low) cells. The Rac1(high) cells invaded more efficiently than did Rac1(low) cells in a Matrigel invasion assay. We assessed the transcriptional profiles of Rac1(high) and Rac1(low) cells and performed gene ontology analysis. Among the 14 genes that were most associated with the term 'membrane' (membrane-related genes) in Rac1(high) cells, we identified four genes that were associated with glioma invasion and Rac1 activity by using siRNA knockdown experiments. Among the transcription factors upregulated in Rac1(high) cells, Egr2 was found to positively regulate expression of the four membrane-related invasion-associated genes. The identified signaling network might cause the fluctuations in Rac1 activity and the heterogeneity in the invasive capacity of glioma cells.

In vivo fluorescence resonance energy transfer imaging reveals differential activation of Rho-family GTPases in glioblastoma cell invasion.

Two-photon excitation microscopy was used to visualized two different modes of invasion at perivascular and intraparenchymal regions of rat C6 glioblastoma cells that were orthotopically implanted into rat brains. Probes based on the principle of Förster resonance energy transfer (FRET) further revealed that glioblastoma cells penetrating the brain parenchyma showed higher Rac1 and Cdc42 activities and lower RhoA activity than those advancing in the perivascular regions. This spatial regulation of Rho-family GTPase activities was recapitulated in three-dimensional spheroid invasion assays with rat and human glioblastoma cells, in which multipod glioblastoma cells that invaded the gels and led the other glioblastoma cells exhibited higher Rac1 and Cdc42 activities than the trailing glioblastoma cells. We also studied the Cdc42-specific guanine nucleotide exchange factor Zizimin1 (also known as DOCK9) as a possible contributor to this spatially controlled activation of Rho-family GTPases, because it is known to play an essential role in the extension of neurites. We found that shRNA-mediated knockdown of Zizimin1 inhibited formation of pseudopodia and concomitant invasion of glioblastoma cells both under a 3D culture condition and in vivo. Our results suggest that the difference in the activity balance of Rac1 and Cdc42 versus RhoA determines the mode of glioblastoma invasion and that Zizimin1 contributes to the invasiveness of glioblastoma cells with high Rac1 and Cdc42 activities.

Intracardiac Injection Mouse Model to Study Cancer Cell Dormancy in Brain Metastasis.

Brain metastasis is a highly complex process, and some cancer cells enter a dormant state after extravasation into the brain. The molecular mechanism of dormancy remains largely unknown and is still under intense investigation. Here, we outline a basic approach to generating and analyzing experimental mouse models to study dormant cancer cells in the brain. Cancer cells stably expressing EGFP and firefly luciferase are injected into the left ventricle of athymic nude mice. After confirmation of brain metastasis by bioluminescence imaging, brain slices are prepared and subjected to Ki67 staining. In addition, a methodology for recovering brain metastatic cancer cells from the mouse brain is described, providing technical tips for unraveling the mysteries of cancer cell dormancy in brain metastasis.

Cell Damage Mechanisms during Cryopreservation in a Zwitterion Solution and Its Alleviation by DMSO.

Recently, zwitterions have been proposed as novel cryoprotectants. However, some cells are difficult to cryopreserve using aqueous zwitterion solutions alone. We investigated here the reason for cell damage in such cells, and it was the osmotic pressure after freeze concentration. Furthermore, the addition of dimethyl sulfoxide (DMSO) has been reported to improve the cryoprotective effect in such cells: the zwitterion/DMSO aqueous solution shows a higher cryoprotective effect than the commercial cryoprotectant. This study also clarified the mechanisms underlying the improvement in a cryoprotective effect. The addition of cell-permeable DMSO alleviated the osmotic pressure after the freeze concentration. This alleviation was also found to be a key factor for cryopreserving cell spheroids, while there has been no insight into this phenomenon.

Optimization of Zwitterionic Polymers for Cell Cryopreservation.

Cryopreservation techniques are valuable for the preservation of genetic properties in cells, and the development of this technology contributes to various fields. In a previous study, an isotonic freezing medium composed of poly(zwitterion) (polyZI) has been reported, which alleviates osmotic shock, unlike typical hypertonic freezing media. In this study, the primitive freezing medium composed of emerging polyZI is optimized. Imidazolium/carboxylate-type polyZI (VimC3C) is the optimal chemical structure. The molecular weight and degree of ion substitution (DSion) are not significant factors. There is an impediment with the primitive polyZI freezing media. While the polyZI forms a matrix around the cell membrane to protect cells, the matrix is difficult to remove after thawing, resulting in low cell proliferation. Unexpectedly, increasing the poly(VimC3C) concentration from 10% to 20% (w/v) improves cell proliferation. The optimized freezing medium, 20% (w/v) poly(VimC3C)_DSion(100%)/1% (w/v) NaCl aqueous solution, exhibited a better cryoprotective effect.

Astrocyte-induced mGluR1 activates human lung cancer brain metastasis via glutamate-dependent stabilization of EGFR.

There are limited methods to stably analyze the interactions between cancer cells and glial cells in vitro, which hinders our molecular understanding. Here, we develop a simple and stable culture method of mouse glial cells, termed mixed-glial culture on/in soft substrate (MGS), which serves well as a platform to study cancer-glia interactions. Using this method, we find that human lung cancer cells become overly dependent on metabotropic glutamate receptor 1 (mGluR1) signaling in the brain microenvironment. Mechanistically, interactions with astrocytes induce mGluR1 in cancer cells through the Wnt-5a/prickle planar cell polarity protein 1 (PRICKLE1)/RE1 silencing transcription factor (REST) axis. Induced mGluR1 directly interacts with and stabilizes the epidermal growth factor receptor (EGFR) in a glutamate-dependent manner, and these cells then become responsive to mGluR1 inhibition. Our results highlight increased dependence on mGluR1 signaling as an adaptive strategy and vulnerability of human lung cancer brain metastasis.

Cryopreservation of tissues by slow-freezing using an emerging zwitterionic cryoprotectant.

Cryopreservation of tissues is a tough challenge. Cryopreservation is categorized into slow-freezing and vitrification, and vitrification has recently been recognized as a suitable method for tissue cryopreservation. On the contrary, some researchers have reported that slow-freezing also has potential for tissue cryopreservation. Although conventional cryoprotectants have been studied well, some novel ones may efficiently cryopreserve tissues via slow-freezing. In this study, we used aqueous solutions of an emerging cryoprotectant, an artificial zwitterion supplemented with a conventional cryoprotectant, dimethyl sulfoxide (DMSO), for cell spheroids. The zwitterion/DMSO aqueous solutions produced a better cryoprotective effect on cell spheroids, which are the smallest units of tissues, compared to that of a commercial cryoprotectant. Cryopreservation with the zwitterion/DMSO solutions not only exhibited better cell recovery but also maintained the functions of the spheroids effectively. The optimized composition of the solution was 10 wt% zwitterion, 15 wt% DMSO, and 75 wt% water. The zwitterion/DMSO solution gave a higher number of living cells for the cryopreservation of mouse tumor tissues than a commercial cryoprotectant. The zwitterion/DMSO solution was also able to cryopreserve human tumor tissue, a patient-derived xenograft.

Cell-compatible isotonic freezing media enabled by thermo-responsive osmolyte-adsorption/exclusion polymer matrices.

During the long-term storage of cells, it is necessary to inhibit ice crystal formation by adding cryoprotectants. Non-cell-permeable cryoprotectants have high osmotic pressure which dehydrates cells, indirectly suppressing intracellular ice crystal formation. However, the high osmotic pressure and dehydration often damage cells. Emerging polymer-type non-cell-permeable cryoprotectants form matrices surrounding cells. These matrices inhibit the influx of extracellular ice nuclei that trigger intracellular ice crystal formation. However, these polymer-type cryoprotectants also require high osmotic pressure to exert an effective cryoprotecting effect. In this study, we designed a poly(zwitterion) (polyZI) that forms firm matrices around cells based on their high affinity to cell membranes. The polyZI successfully cryopreserved freeze-vulnerable cells under isotonic conditions. These matrices also controlled osmotic pressure by adsorbing and desorbing NaCl depending on the temperature, which is a suitable feature for isotonic cryopreservation. Although cell proliferation was delayed by the cellular matrices, washing with a sucrose solution improved proliferation.

Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors.

Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

Stable expression of FRET biosensors: a new light in cancer research.

The constituents of the oncogene signal transduction pathway are promising targets for anticancer drugs. Despite the wealth of available knowledge regarding their molecular properties, the spatiotemporal regulation of the signaling molecules remains elusive. Biosensors based on the principle of FRET have been developed to visualize the activities of the signaling molecules in living cells. However, difficulties in the development of sensitive FRET biosensors have prevented their widespread use in cancer research. The lack of cell lines constitutively expressing a FRET biosensor has also limited their use. In this review, we will introduce the principle of FRET-based biosensors, describe an optimized backbone of the FRET biosensors, techniques to express FRET biosensors stably in the cells, and discuss the future perspectives of FRET biosensors in cancer research.

High loading of trimethylglycine promotes aqueous solubility of poorly water-soluble cisplatin.

Trimethylglycine (TMG) is a cheap, natural, and highly biocompatible compound. Therefore, it has been used in the fields of food and life sciences, but the application of solid TMG is limited to utilisation as an "additive". In the present study, we focussed on the high solubility of TMG in water, derived from the aprotic zwitterionic structure, and proposed TMG as the chemical accounting for a major portion of the aqueous solution (e.g., 50 wt%). High loading of TMG shifted the properties of water and enabled the dissolution of poorly water-soluble cisplatin, an anticancer agent, at high concentration (solubility of cisplatin: 0.15 wt% in water vs 1.7 wt% in TMG aqueous solution). For hepatic arterial infusion, this can reduce the amount of cisplatin administered from 40 to 4 mL. It enables simple injection using a syringe, without the need for catheters and automatic pumps, leading to critical alleviation of the risk to patients. Furthermore, we produced a dry powder from a cisplatin-containing TMG aqueous solution via freeze-drying. Powders can be conveniently stored and transported. Furthermore, cisplatin is often used as a mixture with other drugs, and cisplatin aqueous solutions are not preferred as they dilute the other drugs.

Synthetic zwitterions as efficient non-permeable cryoprotectants.

Cryopreservation of cells is necessary for long periods of storage. However, some cell lines cannot be efficiently cryopreserved, even when optimized commercial cryoprotectants are employed. Previously, we found that a low-toxic synthetic zwitterion aqueous solution enabled good cryopreservation. However, this zwitterion solution could not cryopreserve some cells, such as human kidney BOSC cells, with good efficiency. Therefore, details of the cryoprotective effect of the zwitterions and optimization based on its mechanisms are required. Herein, we synthesized 18 zwitterion species and assessed the effects of the physical properties of water/zwitterion mixtures. Non-cell-permeable zwitterions can inhibit ice crystal formation extracellularly via direct interaction with water and intracellularly via dehydration of cells. However, cells that could not be cryopreserved by zwitterions were insufficiently dehydrated in the zwitterion solution. Dimethyl sulfoxide (DMSO) was combined as a cell-permeable cryoprotectant to compensate for the shortcomings of non-cell-permeable zwitterions. The water/zwitterion/DMSO (90/10/15, v/w/w) could cryopreserve different cells, for example freezing-vulnerable K562 and OVMANA cells; yielding ~1.8-fold cell viability compared to the case using a commercial cryoprotectant. Furthermore, molecular dynamics simulation indicated that the zwitterions protected the cell membrane from the collapse induced by DMSO.

Glioma Stem-Like Cells Can Be Targeted in Boron Neutron Capture Therapy with Boronophenylalanine.

As glioma stem cells are chemo- and radio-resistant, they could be the origins of recurrent malignant glioma. Boron neutron capture therapy (BNCT) is a tumor-selective particle radiation therapy. 10B(n,α)7Li capture reaction produces alpha particles whose short paths (5-9 µm) lead to selective killing of tumor cells. P-boronophenylalanine (BPA) is a chemical compound used in clinical trials for BNCT. Here, we used mass cytometry (Cytof) to investigate whether glioma stem-like cells (GSLCs) take up BPA or not. We used GSLCs, and cells differentiated from GSLCs (DCs) by fetal bovine serum. After exposure to BPA for 24 h at 25 ppm in 5% CO2 incubator, we immune-stained them with twenty stem cell markers, anti-Ki-67, anti-BPA and anti-CD98 (heterodimer that forms the large BPA transporter) antibodies and analyzed them with Cytof. The percentage of BPA+ or CD98+ cells with stem cell markers (Oct3/4, Nestin, SOX2, Musashi-1, PDGFRα, Notch2, Nanog, STAT3 and C-myc, among others) was 2-4 times larger among GSLCs than among DCs. Analyses of in vivo orthotopic tumor also indicated that 100% of SOX2+ or Nestin+ GSLCs were BPA+, whereas only 36.9% of glial fibrillary acidic protein (GFAP)+ DCs were BPA+. Therefore, GSLCs may take up BPA and could be targeted by BNCT.

Non-aqueous, zwitterionic solvent as an alternative for dimethyl sulfoxide in the life sciences.

Dimethyl sulfoxide (DMSO) is widely used as a solvent in the life sciences, however, it is somewhat toxic and affects cell behaviours in a range of ways. Here, we propose a zwitterionic liquid (ZIL), a zwitterion-type ionic liquid containing histidine-like module, as a new alternative to DMSO. ZIL is not cell permeable, less toxic to cells and tissues, and has great potential as a vehicle for various hydrophobic drugs. Notably, ZIL can serve as a solvent for stock solutions of platinating agents, whose anticancer effects are completely abolished by dissolution in DMSO. Furthermore, ZIL possesses suitable affinity to the plasma membrane and acts as a cryoprotectant. Our results suggest that ZIL is a potent, multifunctional and biocompatible solvent that compensates for many shortcomings of DMSO.