Deep Learning of Phase-Contrast Images of Cancer Stem Cells Using a Selected Dataset of High Accuracy Value Using Conditional Generative Adversarial Networks.

Artificial intelligence (AI) technology for image recognition has the potential to identify cancer stem cells (CSCs) in cultures and tissues. CSCs play an important role in the development and relapse of tumors. Although the characteristics of CSCs have been extensively studied, their morphological features remain elusive. The attempt to obtain an AI model identifying CSCs in culture showed the importance of images from spatially and temporally grown cultures of CSCs for deep learning to improve accuracy, but was insufficient. This study aimed to identify a process that is significantly efficient in increasing the accuracy values of the AI model output for predicting CSCs from phase-contrast images. An AI model of conditional generative adversarial network (CGAN) image translation for CSC identification predicted CSCs with various accuracy levels, and convolutional neural network classification of CSC phase-contrast images showed variation in the images. The accuracy of the AI model of CGAN image translation was increased by the AI model built by deep learning of selected CSC images with high accuracy previously calculated by another AI model. The workflow of building an AI model based on CGAN image translation could be useful for the AI prediction of CSCs.

Use of DNA-generated gold nanoparticles to radiosensitize and eradicate radioresistant glioma stem cells.

The surface reactivity of gold nanoparticles (AuNPs) is receiving attention as a radiosensitizer of cancer cells for radiation therapy and/or as a drug carrier to target cells. This study demonstrates the potential of DNA-AuNPs (prepared by mixing calf thymus DNA with HAuCl4 solution) as a radiosensitizer of human glioma cells that have cancer stem cell (CSC)-like properties, to reduce their survival. CSC-like U251MG-P1 cells and their parental glioblastoma U251MG cells are treated with a prepared DNA-AuNP colloid. The radiosensitivity of the resultant AuNP-associated cells are significantly enhanced. To reveal the mechanism by which survival is reduced, the generation of reactive oxygen species (ROS), apoptosis induction, or DNA damage in the cells is assayed using the fluorescent dye DCFDA, annexin V-FITC/PI, and foci formation of γ-H2AX, respectively. X-ray irradiation with administration of AuNPs overcomes the radioresistance of U251MG-P1 cells. It does not induce ROS generation or apoptosis in the cells but enhances the number of abnormal nuclei with abundant γ-H2AX foci, which is judged as cell death by mitotic catastrophe. The AuNP association with the cells effectively induces mitotic catastrophe in x-ray-irradiated CSC-like cells, implicating that DNA-AuNPs might be a promising tool to develop an efficient radiosensitizer against CSC.

HSP-enriched properties of extracellular vesicles involve survival of metastatic oral cancer cells.

Cancer cells often secrete extracellular vesicles (EVs) that carry heat shock proteins (HSPs) with roles in tumor progression. Oral squamous cell carcinoma (OSCC) belongs to head and neck cancers (HNC) whose lymph-node-metastases often lead to poor prognosis. We have examined the EV proteome of OSCC cells and found abundant secretion of HSP90-enriched EVs in lymph-node-metastatic OSCC cells. Double knockdown of HSP90α and HSP90ß, using small interfering RNA significantly reduced the survival of the metastatic OSCC cells, although single knockdown of each HSP90 was ineffective. Elevated expression of these HSP90 family members was found to correlate with poor prognosis of HNC cases. Thus, elevated HSP90 levels in secreted vesicles are potential prognostic biomarkers and therapeutic targets in metastatic OSCC.

Targeting Glioblastoma Cells Expressing CD44 with Liposomes Encapsulating Doxorubicin and Displaying Chlorotoxin-IgG Fc Fusion Protein.

We recently have established a successful xenograft model of human glioblastoma cells by enriching hyaluronic acid-dependent spheroid-forming populations termed U251MG-P1 cells from U251MG cells. Since U251MG-P1 cells have been confirmed to express CD44 along with principal stemness marker genes, OCT3/4, SOX2, KLF4 and Nanog, this CD44 expressing population appeared to majorly consist of undifferentiated cells. Evaluating the sensitivity to anti-cancer agents, we found U251MG-P1 cells were sensitive to doxorubicin with IC50 at 200 nM. Although doxorubicin has serious side-effects, establishment of an efficient therapy targeting undifferentiated glioblastoma cell population is necessary. We previously designed a chlorotoxin peptide fused to human IgG Fc region without hinge sequence (M-CTX-Fc), which exhibited a stronger growth inhibitory effect on the glioblastoma cell line A172 than an original chlorotoxin peptide. Combining these results together, we designed M-CTX-Fc conjugated liposomes encapsulating doxorubicin and used U251MG-P1 cells as the target model in this study. The liposome modified with M-CTX-Fc was designed with a diameter of approximately 100-150 nm and showed high encapsulation efficiency, adequate loading capacity of anticancer drug, enhanced antitumor effects demonstrating increasing uptake into the cells in vitro; M-CTX-Fc-L-Dox shows great promise in its ability to suppress tumor growth in vivo and it could serve as a template for targeted delivery of other therapeutics.

Evaluation of glycosylated docetaxel-encapsulated liposomes prepared by remote loading under solubility gradient.

Docetaxel comprises one of the most effective anti-cancer drugs despite of serious side effects. Liposomes encapsulation is practically feasible to deliver the drug. However, due to the significant hydrophobicity, docetaxel will be integrated into the lipid bilayer resulting in poor encapsulation capacity. Here, we evaluated a remote loading strategy using a solubility gradient made between the two solvents for 7-glucosyloxyacetyldocetaxel, which has enhanced water solubility of docetaxel with a coupled glucose moiety. Therefore, 7-glucosyloxyacetyldocetaxel was more effectively encapsulated into liposomes with 71.0% of encapsulation efficiency than docetaxel. While 7-glucosyloxyacetyldocetaxel exhibited 90.9% of tubulin stabilisation activity of docetaxel, 7-glucosyloxyacetyldocetaxel encapsulated in liposomes significantly inhibited the growth of tumour in vivo with side effects less than unencapsulated drug. Collectively, the encapsulation of 7-glucosyloxyacetyldocetaxel into liposomes by remote loading under the solubility gradient is considered to be a promising application to prepare practical drug delivery system.

Cancer stem cells maintain a hierarchy of differentiation by creating their niche.

The self-renewal and differentiation properties of cancer stem cells (CSCs) are regulated and maintained by the CSC niche. However, the mechanism of this maintenance, especially the maintenance contributed by differentiated cancer cells, remains to be fully elucidated. Recently, we have established a model of CSCs, miPS-LLCcm, from mouse induced pluripotent stem cells (miPSCs). In vitro cultured miPS-LLCcm cells were autonomously balanced with stem-like cells and differentiated cells including vascular endothelial cells. Under these conditions, the CSC properties appeared to be stable in the presence of the factor(s) secreted by the differentiated cells. The factor(s) activated Notch signaling and promoted self-renewal of CSCs. In addition, the secreted factor(s) appeared to regulate the differentiation lineage of CSCs. Our results indicate that the differentiated progenies of CSCs containing vascular endothelium play important roles for regulating the CSC's properties. Therefore, miPS-LLCcm cells create their own in vitro niche to maintain themselves in the hierarchy of differentiating CSCs.

The current status and novel advances of boron neutron capture therapy clinical trials.

Boron neutron capture therapy (BNCT) is a treatment method that focuses on improving the cure rate of patients with cancer who are difficult to treat using traditional clinical methods. By utilizing the high neutron absorption cross-section of boron, material rich in boron inside tumor cells can absorb neutrons and release high-energy ions, thereby destroying tumor cells. Owing to the short range of alpha particles, this method can precisely target tumor cells while minimizing the inflicted damage to the surrounding normal tissues, making it a potentially advantageous method for treating tumors. Globally, institutions have progressed in registered clinical trials of BNCT for multiple body parts. This review summarized the current achievements in registered clinical trials, Investigator-initiated clinical trials, aimed to integrate the latest clinical research literature on BNCT and to shed light on future study directions.

Eosinophil cationic protein enhances stabilization of ß-catenin during cardiomyocyte differentiation in P19CL6 embryonal carcinoma cells.

Prior to gastrulation, the Wnt signaling pathway through stabilized ß-catenin enhances the differentiation of mouse ES cell into cardiomyocytes. We have recently shown that cardiomyocyte differentiation is enhanced by eosinophil cationic protein (ECP) through accelerated expression of marker genes of early cardiac differentiation. Furthermore, ECP enhanced the expression of Wnt3a in P19CL6 cells which were stimulated to differentiate into cardiomyocytes by DMSO. Following these findings, we evaluated in this study the potential of ECP to activate the Wnt/ß-catenin signaling pathway during cardiomyocyte differentiation. Analysis by real time qPCR revealed that ECP increased the expression of Frizzled genes such as Frizzled-1, -2, -4 and -10 in P19CL6 cells in the presence of DMSO. The increased expression of those Wnt receptors was found to inhibit the phosphorylation of ß-catenin resulting in the stabilization and translocation of ß-catenin into the nucleus of P19CL6 cells during the early stages of cardiomyocyte differentiation. When assessed for ß-catenin/TCF transcriptional activity with a TCF-luciferase (TOP/FOP) assay, ECP enhanced luciferase activity in P19CL6 cells during 48 h after transfection with TOP/FOP flash reporter in a stoichiometric manner. Collectively, this suggests that ECP can activate a canonical Wnt/ß-catenin signaling pathway by enhancing the stabilization of ß-catenin during cardiomyocyte differentiation.

Plasma membrane-localized transporter for aluminum in rice.

Aluminum (Al) is the most abundant metal in the Earth's crust, but its trivalent ionic form is highly toxic to all organisms at low concentrations. How Al enters cells has not been elucidated in any organisms. Herein, we report a transporter, Nrat1 (Nramp aluminum transporter 1), specific for trivalent Al ion in rice. Nrat1 belongs to the Nramp (natural resistance-associated macrophage protein) family, but shares a low similarity with other Nramp members. When expressed in yeast, Nrat1 transports trivalent Al ion, but not other divalent ions, such as manganese, iron, and cadmium, or the Al-citrate complex. Nrat1 is localized at the plasma membranes of all cells of root tips except epidermal cells. Knockout of Nrat1 resulted in decreased Al uptake, increased Al binding to cell wall, and enhanced Al sensitivity, but did not affect the tolerance to other metals. Expression of Nrat1 is up-regulated by Al in the roots and regulated by a C2H2 zinc finger transcription factor (ART1). We therefore concluded that Nrat1 is a plasma membrane-localized transporter for trivalent Al, which is required for a prior step of final Al detoxification through sequestration of Al into vacuoles.

Eosinophil cationic protein enhances cardiomyocyte differentiation of P19CL6 embryonal carcinoma cells by stimulating the FGF receptor signaling pathway.

We investigated the functional role of eosinophil cationic protein (ECP) in regulating cardiomyogenesis using mouse P19CL6 embryonic carcinoma cells. ECP was confirmed to accelerate the cardiomyocyte differentiation of P19CL6 cells by enhancing the rate and area size of beating of cardiomyocyte and by facilitating the expression of cardiomyocyte-specific genes, such as GATA4 and α-MHC. Since cardiomyocyte differentiation in vivo is considered to follow mesoderm induction, the induction of Brachyury, a marker of mesoderm, was assessed. Brachyury expression was found to be enhanced after the addition of ECP. This enhancement was due to the stimulation of extracellular signal-regulated kinase (ERK)1/2 phosphorylation by ECP. In this context, treatment with SU5402, an inhibitor of fibroblast growth factor (FGF) receptor 1, suppressed Brachyury expression, phosphorylation of ERK1/2, and cardiomyocyte differentiation induced by ECP. We concluded that ECP might induce mesoderm differentiation through FGF signaling pathway and enhance subsequent cardiomyocyte differentiation in concert with dimethyl sulfoxide in P19CL6 cells. ECP may be a novel factor for cardiomyocyte differentiation, which should be very useful to prepare adequate numbers of cardiomyocytes for therapeutic cell transplantation.

The conformational polymorphism of the green fluorescent protein.

Green fluorescent protein (GFPuv) has been widely used as a reporter fused to individual targeting sequences. However, its state in liquid and its effect on other proteins are still unclear. The conformational polymorphisms of glutathione-S-transferase-green fluorescent protein (GST-GFPuv), GFPuv and GST were analyzed by native polyacrylamide gel, indicating that GST was in many different states while GFPuv and GST-GFPuv were only in four and two slightly different states. Four different circular dichroism spectra were obtained from the GFPuv polymorphisms. The single molecular behavior of GST-GFPuv and GFPuv was also characterized by MALDI-TOF MS. Thus, we demonstrated that: (1) there might be four different structural polymorphisms for the native GFPuv; (2) GFPuv could reduce its partner's polymorphism as a fusion protein. Although GFPuv had many merits as a reporter, its unreliability was found in the study.

Temporal and Locational Values of Images Affecting the Deep Learning of Cancer Stem Cell Morphology.

Deep learning is being increasingly applied for obtaining digital microscopy image data of cells. Well-defined annotated cell images have contributed to the development of the technology. Cell morphology is an inherent characteristic of each cell type. Moreover, the morphology of a cell changes during its lifetime because of cellular activity. Artificial intelligence (AI) capable of recognizing a mouse-induced pluripotent stem (miPS) cell cultured in a medium containing Lewis lung cancer (LLC) cell culture-conditioned medium (cm), miPS-LLCcm cell, which is a cancer stem cell (CSC) derived from miPS cell, would be suitable for basic and applied science. This study aims to clarify the limitation of AI models constructed using different datasets and the versatility improvement of AI models. The trained AI was used to segment CSC in phase-contrast images using conditional generative adversarial networks (CGAN). The dataset included blank cell images that were used for training the AI but they did not affect the quality of predicting CSC in phase contrast images compared with the dataset without the blank cell images. AI models trained using images of 1-day culture could predict CSC in images of 2-day culture; however, the quality of the CSC prediction was reduced. Convolutional neural network (CNN) classification indicated that miPS-LLCcm cell image classification was done based on cultivation day. By using a dataset that included images of each cell culture day, the prediction of CSC remains to be improved. This is useful because cells do not change the characteristics of stem cells owing to stem cell marker expression, even if the cell morphology changes during culture.

Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand.

Targeting and down-regulation of ErbB2, a member of EGF receptor family, is regarded as one of the key aspect for cancer treatment because it is often overexpressed in breast and ovarian cancer cells. Although natural ligands for ErbB2 have not been found, unlike other ErbB receptors, EC-1, a 20-amino acid circular peptide, has been shown to bind to ErbB2 as an artificial ligand. Previously we showed EC-1 peptide did not induce the internalization of ErbB2 in SK-BR-3 cells. In this report, we designed divalent and multivalent forms of EC-1 peptide with the Fc portion of the human IgG and bionanocapsule modified with ZZ-tag on its surface to improve the interaction with ErbB2. These forms showed higher affinity to ErbB2 than that of EC-1 monomer. Furthermore, prominent endosomal accumulation of ErbB2 occurred in SK-BR-3 cells when stimulated with EC-Fc ligand multivalently displayed on the surface of the bionanocapsule, whereas SK-BR-3 cells as themselves displayed stringent mechanism against ErbB2 internalization without stimulation. The multivalent form of EC-1 peptide appeared to internalize ErbB2 more efficiently than divalent form did. This internalization was unaffected by the inhibition of clathrin association, but inhibited when the cholesterol was depleted which explained either caveolar or GPI-AP-early endocytic compartment (GEEC) pathway. Because of the lack of caveolin-1 expression, caveolar machinery may be lost in SK-BR-3 cell line. Therefore, it is suggested that the multivalent form of EC-1 induces the internalization of ErbB2 through the GEEC pathway.

Structural and spatial associations between Fe, O, and C in the network structure of the Leptothrix ochracea sheath surface.

The structural and spatial associations of Fe with O and C in the outer coat fibers of the Leptothrix ochracea sheath were shown to be substantially similar to the stalk fibers of Gallionella ferruginea, i.e., a central C core, probably of bacterial origin, and aquatic Fe interacting with O at the surface of the core.

Isolation of a Leptothrix strain, OUMS1, from ocherous deposits in groundwater.

Leptothrix species in aquatic environments produce uniquely shaped hollow microtubules composed of aquatic inorganic and bacterium-derived organic hybrids. Our group termed this biologically derived iron oxide as "biogenous iron oxide (BIOX)". The artificial synthesis of most industrial iron oxides requires massive energy and is costly while BIOX from natural environments is energy and cost effective. The BIOX microtubules could potentially be used as novel industrial functional resources for catalysts, adsorbents and pigments, among others if effective and efficient applications are developed. For these purposes, a reproducible system to regulate bacteria and their BIOX productivity must be established to supply a sufficient amount of BIOX upon industrial demand. However, the bacterial species and the mechanism of BIOX microtubule formation are currently poorly understood. In this study, a novel Leptothrix sp. strain designated OUMS1 was successfully isolated from ocherous deposits in groundwater by testing various culture media and conditions. Morphological and physiological characters and elemental composition were compared with those of the known strain L. cholodnii SP-6 and the differences between these two strains were shown. The successful isolation of OUMS1 led us to establish a basic system to accumulate biological knowledge of Leptothrix and to promote the understanding of the mechanism of microtubule formation. Additional geochemical studies of the OUMS1-related microstructures are expected provide an attractive approach to study the broad industrial application of bacteria-derived iron oxides.

Self-assembling A6K peptide nanotubes as a mercaptoundecahydrododecaborate (BSH) delivery system for boron neutron capture therapy (BNCT).

Boron neutron capture therapy (BNCT) is a tumor selective therapy, the effectiveness of which depends on sufficient 10B delivery to and accumulation in tumors. In this study, we used self-assembling A6K peptide nanotubes as boron carriers and prepared new boron agents by simple mixing of A6K and BSH. BSH has been used to treat malignant glioma patients in clinical trials and its drug safety and availability have been confirmed; however, its contribution to BNCT efficacy is low. A6K nanotube delivery improved two major limitations of BSH, including absence of intracellular transduction and non-specific drug delivery to tumor tissue. Varying the A6K peptide and BSH mixture ratio produced materials with different morphologies-determined by electron microscopy-and intracellular transduction efficiencies. We investigated the A6K/BSH 1:10 mixture ratio and found high intracellular boron uptake with no toxicity. Microscopy observation showed intracellular localization of A6K/BSH in the perinuclear region and endosome in human glioma cells. The intracellular boron concentration using A6K/BSH was almost 10 times higher than that of BSH. The systematic administration of A6K/BSH via mouse tail vein showed tumor specific accumulation in a mouse brain tumor model with immunohistochemistry and pharmacokinetic study. Neutron irradiation of glioma cells treated with A6K/BSH showed the inhibition of cell proliferation in a colony formation assay. Boron delivery using A6K peptide provides a unique and simple strategy for next generation BNCT drugs.

Deep Learning of Cancer Stem Cell Morphology Using Conditional Generative Adversarial Networks.

Deep-learning workflows of microscopic image analysis are sufficient for handling the contextual variations because they employ biological samples and have numerous tasks. The use of well-defined annotated images is important for the workflow. Cancer stem cells (CSCs) are identified by specific cell markers. These CSCs were extensively characterized by the stem cell (SC)-like gene expression and proliferation mechanisms for the development of tumors. In contrast, the morphological characterization remains elusive. This study aims to investigate the segmentation of CSCs in phase contrast imaging using conditional generative adversarial networks (CGAN). Artificial intelligence (AI) was trained using fluorescence images of the Nanog-Green fluorescence protein, the expression of which was maintained in CSCs, and the phase contrast images. The AI model segmented the CSC region in the phase contrast image of the CSC cultures and tumor model. By selecting images for training, several values for measuring segmentation quality increased. Moreover, nucleus fluorescence overlaid-phase contrast was effective for increasing the values. We show the possibility of mapping CSC morphology to the condition of undifferentiation using deep-learning CGAN workflows.

A Novel Combination Cancer Therapy with Iron Chelator Targeting Cancer Stem Cells via Suppressing Stemness.

Excess iron causes cancer and is thought to be related to carcinogenesis and cancer progression including stemness, but the details remain unclear. Here, we hypothesized that stemness in cancer is related to iron metabolism and that regulating iron metabolism in cancer stem cells (CSCs) may be a novel therapy. In this study, we used murine induced pluripotent stem cells that expressed specific stem cell genes such as Nanog, Oct3/4, Sox2, Klf4, and c-Myc, and two human cancer cell lines with similar stem cell gene expression. Deferasirox, an orally available iron chelator, suppressed expression of stemness markers and spherogenesis of cells with high stemness status in vitro. Combination therapy had a marked antitumor effect compared with deferasirox or cisplatin alone. Iron metabolism appears important for maintenance of stemness in CSCs. An iron chelator combined with chemotherapy may be a novel approach via suppressing stemness for CSC targeted therapy.

Up-Regulation of PI 3-Kinases and the Activation of PI3K-Akt Signaling Pathway in Cancer Stem-Like Cells Through DNA Hypomethylation Mediated by the Cancer Microenvironment.

Previously, we have succeeded in converting induced pluripotent stem cells (iPSCs) into cancer stem cells (CSCs) by treating the iPSCs with conditioned medium of Lewis lung carcinoma (LLC) cells. The converted CSCs, named miPS-LLCcm cells, exhibited the self-renewal, differentiation potential, and potential to form malignant tumors with metastasis. In this study, we further characterized miPS-LLCcm cells both in vivo and in vitro. The tumors formed by subcutaneous injection showed the structures with pathophysiological features consisting of undifferentiated and malignant phenotypes generally found in adenocarcinoma. Metastasis in the lung was also observed as nodule structures. Excising from the tumors, primary cultured cells from the tumor and the nodule showed self-renewal, differentiation potential as well as tumor forming ability, which are the essential characters of CSCs. We then characterized the epigenetic regulation occurring in the CSCs. By comparing the DNA methylation level of CG rich regions, the differentially methylated regions (DMRs) were evaluated in all stages of CSCs when compared with the parental iPSCs. In DMRs, hypomethylation was found superior to hypermethylation in the miPS-LLCcm cells and its derivatives. The hypo- and hypermethylated genes were used to nominate KEGG pathways related with CSC. As a result, several categories were defined in the KEGG pathways from which most related with cancers, significant and high expression of components was PI3K-AKT signaling pathway. Simultaneously, the AKT activation was also confirmed in the CSCs. The PI3K-Akt signaling pathway should be an important pathway for the CSCs established by the treatment with conditioned medium of LLC cells.

Suppression effect on IFN-γ of adipose tissue-derived mesenchymal stem cells isolated from ß2-microglobulin-deficient mice.

Administration of bone marrow-derived mesenchymal stem cells (MSCs) is a possible treatment for graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation and other inflammatory conditions. To address the mechanism of immunosuppression by MSCs, in particular those derived from adipose tissue (AMSCs), AMSCs were isolated from three different mouse strains, and the suppressive capacity of the AMSCs thus obtained to suppress interferon (IFN)-γ generation in mixed lymphocyte reaction cultures serving as an in vitro model of GVHD were assessed. It was revealed that the AMSCs had a potent capacity to suppress IFN-γ production regardless of their strain of origin and that such suppression was not associated with production of interleukin-10. In addition, the results demonstrated that ß2-microglobulin (ß2m)-deficient AMSCs from ß2m-/- mice were also potent suppressor cells, verifying the fact that the mechanism underlying the suppression by AMSCs is independent of major histocompatibility complex (MHC) class I expression or MHC compatibility. As AMSCs appear to have immunosuppressive properties, AMSCs may be a useful source of biological suppressor cells for the control of GVHD in humans.