Publications

Recent Publications

Article

Mammary cell gene expression atlas links epithelial cell remodeling events to breast carcinogenesis

The female mammary epithelium undergoes reorganization during development, pregnancy, and menopause, linking higher risk with breast cancer development. To characterize these periods of complex remodeling, here we report integrated 50 K mouse and 24 K human mammary epithelial cell atlases obtained by single-cell RNA sequencing, which covers most lifetime stages. Our results indicate a putative trajectory that originates from embryonic mammary stem cells which differentiates into three epithelial lineages (basal, luminal hormone- sensing, and luminal alveolar), presumably arising from unipotent progenitors in postnatal glands. The lineage-specific genes infer cells of origin of breast cancer using The Cancer Genome Atlas data and single-cell RNA sequencing of human breast cancer, as well as the association of gland reorganization to different breast cancer subtypes. This comprehensive mammary cell gene expression atlas (https://mouse-mammary-epitheliumintegrated. cells.ucsc.edu) presents insights into the impact of the internal and external stimuli on the mammary epithelium at an advanced resolution.

Article

Mammary cell gene expression atlas links epithelial cell remodeling events to breast carcinogenesis

Supplementary

Cell3iMager Estier

Brochure

Cell3imager Estier

3D Live-cell imaging with near infrared using Optical Coherence Tomography.

Publication - Journal of Materials Chemistry B

A 3D invitro supported microvessel model:

Angiogenesis, which refers to the formation of new blood vessels from already existing vessels, is a promising therapeutic target and a complex multistep process involving many different factors. Pericytes (PCs) are attracting attention as they are considered to make significant contributions to the maturation and stabilisation of newly formed vessels, although not much is known about the precise mechanisms involved. Since there is no single specific marker for pericytes, in vivo models may complicate PC identification and the study of PCs in angiogenesis would benefit from in vitro models recapitulating the interactions between PCs and endothelial cells (ECs) in a three-dimensional (3D) configuration. In this study, a 3D in vitro co-culture microvessel model incorporating ECs and PCs was constructed by bottom-up tissue engineering. Angiogenesis was induced in the manner of sprout formation by the addition of a vascular endothelial cell growth factor. It was found that the incorporation of PCs prevented expansion of the parent vessel diameter and enhanced sprout formation and elongation. Physical interactions between ECs and PCs were visualised by immunostaining and it disclosed that PCs covered the EC monolayer from its basal side in the parent vessel as well as angiogenic sprouts. Furthermore, the microvessels were visualized in 3D by using a non-invasive optical coherence tomography (OCT) imaging system and sprout features were quantitatively assessed. It revealed that the sprouts in EC–PC co-culture vessels were longer and tighter than those in EC mono-culture vessels. The combination of the microvessel model and the OCT system analysis can be useful for the visualisation and demonstration of the multistep process of angiogenesis, which incorporates PCs.

Publication - Nature/Scientific Report

Directed differentiation of human induced pluripotent stem cells into mature stratified bladder urothelium

For augmentation or reconstruction of urinary bladder after cystectomy, bladder urothelium derived from human induced pluripotent stem cells (hiPSCs) has recently received focus. However, previous studies have only shown the emergence of cells expressing some urothelial markers among derivatives of hiPSCs, and no report has demonstrated the stratified structure, which is a particularly important attribute of the barrier function of mature bladder urothelium. In present study, we developed a method for the directed differentiation of hiPSCs into mature stratified bladder urothelium. The caudal hindgut, from which the bladder urothelium develops, was predominantly induced via the high-dose administration of CHIR99021 during definitive endoderm induction, and this treatment subsequently increased the expressions of uroplakins. Terminal differentiation, characterized by the increased expression of uroplakins, CK13, and CK20, was induced with the combination of Troglitazone + PD153035. FGF10 enhanced the expression of uroplakins and the stratification of the epithelium, and the transwell culture system further enhanced such stratification. Furthermore, the barrier function of our urothelium was demonstrated by a permeability assay using FITC-dextran. According to an immunohistological analysis, the stratified uroplakin II-positive epithelium was observed in the transwells. This method might be useful in the field of regenerative medicine of the bladder.

Publication - Biomaterials

EGFL7 regulates sprouting angiogenesis and endothelial integrity in a human blood vessel model

Elucidating the mechanisms underlying sprouting angiogenesis and permeability should enable the developmentof more effective therapies for various diseases, including retinopathy, cancer, and other vascular disorders. Wefocused on epidermal growth factor-like domain 7 (EGFL7) which plays an important role in NOTCH signalingand in the organization of angiogenic sprouts. We developed anEGFL7-knockdownin vitromicrovessel modeland investigated the effect of EGFL7 at a tissue level. We foundEGFL7knockdown suppressed VEGF-A-inducedsprouting angiogenesis accompanied by an overproduction of endothelialfilopodia and reduced collagen IVdeposition at the basal side of endothelial cells. We also observed impaired barrier function which reflected aninflammatory condition. Furthermore, our results showed that proper formation of adherens junctions andphosphorylation of VE-cadherin was disturbed. In conclusion, by using a 3D microvessel model we identifiednovel roles for EGFL7 in endothelial function during sprouting angiogenesis.

Publication - EBioMedecine

A vascular endothelial growth factor-dependent sprouting angiogenesis assay based on an In vitro human blood vessel model for the study of anti-angiogenic drugs

Angiogenesis is the formation of new capillaries from pre-existing blood vessels and participates in proper vas-culature development. In pathological conditions such as cancer, abnormal angiogenesis takes place. Angiogen-esis is primarily carried out by endothelial cells, the innermost layer of blood vessels. The vascular endothelialgrowth factor-A (VEGF-A) and its receptor-2 (VEGFR-2) trigger most of the mechanisms activating and regulat-ing angiogenesis, and have been the targets for the development of drugs. However, most experimental assaysassessing angiogenesis rely on animal models. We report anin vitromodel using a microvessel-on-a-chip. Itmimics an effective endothelial sprouting angiogenesis event triggered from an initial microvessel using a singleangiogenic factor, VEGF-A. The angiogenic sprouting in this model is depends on the Notch signaling, as observedin vivo. This model enables the study of anti-angiogenic drugs which target a specific factor/receptor pathway, asdemonstrated by the use of the clinically approved sorafenib and sunitinib for targeting the VEGF-A/VEGFR-2pathway. Furthermore, this model allows testing simultaneously angiogenesis and permeability. It demonstratesthat sorafenib impairs the endothelial barrier function, while sunitinib does not. Suchin vitrohuman model pro-vides a significant complimentary approach to animal models for the development of effective therapies.

Publication - Biomaterials Science

A simple three-dimensional gut model constructed in a restricted ductal microspace induces intestinal epithelial cell integrity and facilitates absorption assays

The intestine acts as a center for nutrient and water absorption at the epithelium and plays an important role in immunity. Considering the complexity of its function and roles in living systems, a physiologically relevant gut in vitro model is desirable in both basic biology and the analysis of effects of some substances on functions of the gut; these analyses include the screening of drug and food candidates with regard to intestinal disorder at an early stage of medical development. In the present study, we constructed a three-dimensional (3D) gut model using human absorptive enterocytes (CACO-2 cells) by reconstitution of the gut epithelial sheet restricted on a high-reproducible ductal scaffold of collagen gel. Moreover, using the 3D gut model, we evaluated the morphology at cellular and tissue levels and conducted a phenotypic analysis of the intestinal physiological functions, which involved a permeability assay mimicking barrier disruption inducing inflammation and an absorption assay reflecting ingestive effects. The ductal structure, in vivo-like 3D epithelial structures, epithelial barrier, and effective absorptive function characterized the 3D gut model. The epithelial cells formed a villus-like buckling epithelium, vertical microvilli of increased density on the cell surface, and a crypt-like localized cell proliferating region. The mature shape of the epithelium may contribute to mimicking barrier function and effective absorption compared with that in the 2D gut model. Furthermore, we successfully mimicked the dextran sodium sulfate-induced epithelial barrier dysfunction as a trigger phenomenon of gut inflammation in the 3D gut model. The integrity of the epithelium and phenotypic analysis of the intestinal physiological functions in the simple and reproducible 3D gut model will allow for a drug screening system for assessing the effects on the functions of the gut epithelium from the lumen side.

Publication - Nature/Scientific Report

Visualizing dynamics of angiogenic sprouting from a three-dimensional microvasculature model

Three-dimensional (3D) in vitro microvasculature in a polydimethylsiloxane-based microdevice was developed as a physiologically relevant model of angiogenesis. The angiogenic process is monitored using stage-top optical coherence tomography (OCT). OCT allows non-invasive monitoring of the 3D structures of the prepared host microvasculature and sprouted neovasculature without fluorescence staining. OCT monitoring takes only a few minutes to scan through the several-millimetre scale range, which provides the advantage of rapid observation of living samples. The obtained OCT cross-sectional images capture 3D features of the angiogenic sprouting process and provide information on the dynamics of luminal formation. The stage-top system used in this study enables the observer to visualize the in vitro dynamics of 3D cultured cells simply and conveniently, offering an alternative monitoring method for studies on angiogenesis and providing quantitative information about vascular morphological changes.

Poster

Evaluation method of skin model thickness from 3D image by OCT

As a skin model, sheet like epidermal tissues cultured on insert well are widely used in assays Thickness of the skin sheets is known as one of these phenotypes, and it is effective feature for capturing morphological changes As a method for measuring the thickness, it is common to prepare a tissue section by the paraffin embedding method and measure the length from the upper part to the lower part of the tissue with a microscope However, since this method is invasive evaluation method, subsequent detailed assay cannot be performed In addition, because the evaluation is based on a tissue section, it is a local evaluation, and it is difficult to capture features such as distribution of tissue thickness Furthermore, embedding with paraffin is difficult to maintain the original shape of the tissue Therefore, in order to perform more appropriate thickness evaluation, a new method for non invasively observing and quantifying the tissue on the insert well is required In recent years, optical coherence tomography (OCT) has become widespread as a three dimensional diagnostic method for the fund us. OCT is a technique for rendering a three dimensional tissue as a tomographic image, and the near infrared light, used by OCT as a light source, is suitable for biological permeabili ty and low in cytotoxicity. In this study, the OCT imaging device Cell3iMager Estier (SCREEN Holdings Co., Ltd.) was used to acquire 3D images of non invasively stacked cell tissues, and we aimed to establish a n ew method for measuring of tissue thickness distribution. The cell sheet on the insert well was observed using Cell3iMager Estier , and a three dimensional image was obtained. Based on the obtained three dimensional image, cell regions were extracted by image processing, and the thickness distribution of the entire sheet was ca lcu lated. We report that it is possible to measure non invasively the change of the thickness distribution of the whole cell sheet by the experimental condition.

Poster-

Novel OCT technology for non-invasive 3D imaging

3 D ex vivo platforms are being diligently evaluated as better predictive drug efficacy testing tools in preclinical as well as clinical space in the quest for profiling of novel anticancer entities (as single or two drug combinations) Within this context, there has been growing need in improved imaging and analysis of complex 3 D structures Optical Coherent tomography ( has been widely used as one of the most important test in ophthalmology It is a non invasive imaging technology that renders the high resolution and cross sectional images from retina Given its tremendous use in in vivo application, in recently, the technology has been applied in 3 D in vitro ex vivo applications for performing imaging of spheroids/ organoids and large tissues This technology allows to perform l arge tissue imagin g, non invasive monitoring of macro and sprouted neo vasculature without the need for fluorescent staining for providing quantitative information about the vascular morphological changes, thereby allowing for the evaluation of anti angiogenic drugs in real time Aim To develop a novel OCT based technology for imaging and analysis of 3 D structures, such as, spheroid / tissues for g rowth morphological evaluation, q uantification of internal cavities d rug sensitivity t esting to capture the events leading to tumor cell death and assessing effect of anti angiogenic drugs Material Methods and Results All cells were cultured in a suitable manner, and cells were imaged by spectrum domain OCT (SD OCT) system The SD OCT system is outlined below The samples were imaged from the bottom surface Original images obtained by the OCT system contained noise (such as from the collagen gel surrounding the sample) To reduce this noise, image processing was applied for the collected original OCT images using original our software ( and ImageJ image processing software ( as follows The images were subsequently processed with filters The images were then converted into binary images so that the cell area is white and all other areas are black Each feature values ( cavities etc were analyzed

Technology Report- Journal of Reproduction and Development

3D live imaging of Bovine embryos

While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggested that these 22 parameters can be potentially employed to evaluate the quality of bovine before ET.

Cell3iMager Duos 2

Publication- Nature/Science Reports

Ganglioside GM2, highly expressed in the MIA PaCa-2 pancreatic ductal adenocarcinoma cell line, is correlated with growth, invasion, and advanced stage

Gangliosides, a group of glycosphingolipids, are known to be cell surface markers and functional factors in several cancers. However, the association between gangliosides and pancreatic ductal adenocarcinoma (PDAC) has not been well elucidated. In this study, we examined the expression and roles of ganglioside GM2 in PDAC. GM2+ cells showed a higher growth rate than GM2- cells in the adherent cond it io n. When GM2- and GM2+ cells were cultured three-dimensionally, almost all cells in the spheres expressed GM2, including cancer stem cell (CSC)-like cells. A glycolipid synthesis inhibitor reduced GM2 expression and TGF-f31 signaling in these CSC-like cells, presumably by inhibiting the interaction between GM2 and TGF f3 RII and suppressing invasion. Furthermore, suppression ofGM2 expression by MAPK inhibition also reduced TGF-f31 signaling and suppressed invasion. GM2+ cells formed larger subcutaneous tumors at a high incidence in nude mice than did GM2- cells. In PDAC cases, GM2 expression was significantly associated with younger age, larger tumor size, advanced stage and higher histological grade . These findings suggest that GM2 could be used as a novel diagnostic and therapeutic target for PDAC.

Publication- Nature/Science Reports

Hydrodynamic stress stimulates growth of cell clusters via the ANXA1_PI3K_AKT axis in colorectal cancer

Cancer cells are exposed to various stresses in vivo, including hydrodynamic stress (HDS). HDS on cancer cells in the blood stream can influence the metastatic potential. Recent studies revealed that circulating tumor cell clusters are more responsible for metastasis than circulating single cells. Nevertheless, most studies on HDS are based on single cells prepared from established cancer cell lines. Here, we used cancer tissue-originated spheroids (CTOS) as a patient-derived, 3D organoid model to investigate the effect of HDS on cancer cell clusters. We found that HDS induced the growth of cancer cell clusters in a population of colorectal CTOSs. Microarray analyses revealed that the multifunctional protein, Annexin 1 (ANXA1), was upregulated upon HDS exposure. Chemically-induced membrane damage also triggered the expression of ANXA1. A knockdown of ANXA1 revealed that ANXA1 regulated HDS-stimulated growth in colorectal CTOSs. Mechanistically, activating the PI3K/AKT pathway downstream of ANXA1 contributed to the phenotype. These findings demonstrate that HDS induces the growth of cancer cell clusters via ANXA1/PI3K/AKT axis, which helps to elucidate the prometastatic feature of circulating cancer cell clusters.

Poster

Label-free image analysis and its applications

In recent years, research on organoids in regenerative medicine and drug discovery research has developed rapidly. In particular, the research for producing intestinal organoids from induced pluripotent stem cells (iPS cells) and human biological tissues has been remarkably developed, and the culture conditions for each have been almost established. However, imaging and image analysis methods of organoids have not been established yet. Organoids are composed of heterogeneous cell populations, and in addition to exhibiting a non-uniform morphology for each organoid, cell populations other than the evaluation target are likely to be included in the image. For these reasons, only by using image processing with a simple parameter threshold, high-precision segmentation of organoids cannot be performed, and quantification by image analysis is difficult. In the case of image analysis using a fluorescent label, there is a possibility that the above problems can be overcome, but today, the importance of label-free analysis without using a fluorescent label is increasing. In general, a three-dimensional culture method with an extracellular matrix is used for organoid culture, which makes imaging itself and image analysis more difficult. Therefore, in this study, we attempted a bright field image analysis of human iPSC-derived intestinal organoid using deep learning technology, which utilized in various fields. Since individual organoids cultured in Matrigel are located at different heights, all-in-focus images were obtained by Z-stack imaging. By using deep learning on the acquired image, only organoids exhibiting specific morphologies were segmented from the image, and feature quantities such as organoid count, diameter and area were calculated. Cell3iMager duos (SCREEN Holdings Co., Ltd.) was used for this imaging and image analysis. In this presentation, we will discuss the label-free analysis of organoids and their applications

Publication- Current Protocols in Stem Cell Biology

Methods for Automated Single Cell Isolation and Sub-Cloning of Human Pluripotent Stem cells

Advances in human pluripotent stem cell (hPSC) techniques have led them to become a widely used and powerful tool for a vast array of applications, including disease modeling, developmental studies, drug discovery and testing, and emerging cell-based therapies. hPSC workflows that require clonal expansion from single cells, such as CRISPR/Cas9-mediated genome editing, face major challenges in terms of efficiency, cost, and precision. Classical subcloning approaches depend on limiting dilution and manual colony picking, which are both time-consuming and labor-intensive, and lack a real proof of clonality. Here we describe the application of three different automated cell isolation and dispensing devices that can enhance the single-cell cloning process for hPSCs. In combination with optimized cell culture conditions, these devices offer an attractive alternative compared to manual methods. We explore various aspects of each device system and define protocols for their practical application. Following the workflow described here, single cell−derived hPSC sub-clones from each system maintain pluripotency and genetic stability. Furthermore, the workflows can be applied to uncover karyotypic mosaicism prevalent in bulk hPSC cultures. Our robust automated workflow facilitates high-throughput hPSC clonal selection and expansion, urgently needed in the operational pipelines of hPSC applications.

Poster

Novel bone microenvironment model of prostate cancer with chitosan fiber matrix and osteoblast in 3D culture

The interaction between prostate cancer cells and osteoblast is essential for the development of bone metastasis. Recently, novel androgen receptor-axis-targeted agents (ARATs) have been approved for metastatic castration naïve prostate cancer (mCNPC), or non-metastatic castration resistant prostate cancer (nmCRPC), both of which should be pivotal to investigate the association between bone microenvironment and tumor. We established a novel 3D in vitro culture method reflecting bone microenvironment and evaluated the drug susceptibility of ARATs including enzalutamide, apalutamide, darolutamide, abiraterone (Abi) with/without dutasteride (Duta).

Cell 3imager NEO

Publication - PLOS ONE

A novel electron emission-based cell culture device promotes cell proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells

In this report we demonstrate the effect of a novel electron emission-based cell culture device on the proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells. Our device has an electron emission element that allows, for the first time, stable emission of electrons into an atmosphere. Atmospheric electrons react with gas molecules to generate radicals and negative ions, which induce a variety of biochemical reactions in the attached cell culture system. In this study, we demonstrated the effect of this new electron emissionbased cell culture device on cell proliferation and differentiation using pre-osteoblastic MC3T3-E1 cells. Electron emission stimulation (EES) was applied directly to culture medium containing plated cells, after which the number of living cells, the mRNA levels of osteogenesis-related genes, and the alkaline phosphatase (ALP) activity were evaluated. The growth rate of EES-exposed cells increased by approximately 20% in comparison with unexposed control cells. We also found the mRNA levels of osteogenic specific genes such as collagen type I α-1, core-binding factor α-1, and osteocalcin to be up-regulated following EES. ALP activity, a marker for osteogenic activity, was significantly enhanced in EEStreated cells. Furthermore, reactive oxygen species generated by EES were measured to determine their effect on MC3T3-E1 cells. These results suggest that our new electron emission- based cell culture device, while providing a relatively weak stimulus in comparison with atmospheric plasma systems, promotes cell proliferation and differentiation. This system is expected to find application in regenerative medicine, specifically in relation to bone regeneration.

Publication - Molecular Cancer Therapeutics

Acquired JHDM1D BRAF Fusion Confers Resistance to FGFR Inhibition in FGFR2_Amplified Gastric Cancer

FGFR2 gene is frequently amplified in gastric cancer. Recently, targeting FGFR2 has drawn attention as a form of gastric cancer therapy, and FGFR-selective inhibitors have shown promising efficacy in clinical studies. Because overcoming acquired resistance is a common problem with molecular targeting drugs, we investigated a resistant mechanism of FGFR inhibitors using the gastric cancer cell line SNU-16, which harbors FGFR2 amplification. We established single-cell clones of FGFR inhibitor–resistant SNU-16 (AZD-R) by continuous exposure to AZD4547, a selective FGFR inhibitor. To screen the genetic alterations acquired in AZD-R, we ran a comparative genomic hybridization assay and found an amplification of Chr7q34 region. The chromosomal breakpoints were located between the 12th and the 13th exon of jumonji C domain containing histone demethylase 1 homolog D (JHDM1D) and between the 3rd and the 4th exon of BRAF. We sequenced cDNA of the AZD-R clones and found fusion kinase JHDM1D-BRAF, which has previously been identified in primary ovarian cancer. Because JHDM1D–BRAF fusion lacks a RAS-binding domain, the dimerization of JHDM1D–BRAF was enhanced. A cell growth inhibition assay using MEK inhibitors and RAF-dimer inhibitors indicated the dependence of AZD-R clones for growth on the MAPK pathway. Our data provide a clinical rationale for using a MEK or RAF dimer inhibitor to treat FGFR2-amplified gastric cancer patients who have acquired resistance through the JHDN1D–BRAF fusion. Mol Cancer Ther; 17(10); 2217–25. 2018 AACR.

Publication - ETH Doctoral Thesis

3 PREDICTING METABOLISM RELATED DRUG-DRUG INTERACTIONS USING A MULTI TISSUE MICROPHYSIOLOGICAL SYSTEM

This thesis focuses on the design, realization and application of gravity-driven microfluidic systems to create a physiological culture environment for the experimentation with 3D microtissues. As the pharmaceutical industry is facing a prediction dilemma in substance testing, new testing methods are required to bridge the gap between conventional 2D cell culture methods, animal testing, and human physiology. Advanced 3D cell culture techniques in combination with microfluidic technology bear the potential to increase the predictive power of preclinical testing methods and to better characterize the effects of substances on the human organism. However, such systems have yet to be adapted to industrial requirements in terms of material properties, user-friendliness, and experimental throughput. In this thesis, two gravitydriven chip systems and the respective proof-of-concept applications are presented: (i) A scalable, polystyrene-based microfluidic chip was used for combining multiple tissue types in an in vitro system. Primary human liver microtissues and colon cancer microtissues were cultured together and fluidically connected through cell culture medium. Upon co-administration of anticancer therapeutics and other compounds, liver-related drug-drug interactions were detected. (ii) Dynamically changing dosing curves were generated by applying a specific microfluidic channel layout. Colon cancer microtissues were exposed to physiologically-relevant pharmacokinetic dosing curves, and their time-dependent response was assessed. By relying on standardized 3D microtissues and gravity-driven perfusion, the systems presented in this thesis are scalable, robust, and easy to use so that a transfer to industrial settings is conceivable. The overall goal is to devise in vitro systems that closely mimic human physiology.

Publication - Nature/Scientific Reports

Automated high_throughput measurement of size and growth curves of small organisms in well plates

Organism size and growth curves are important biological characteristics. Current methods to measure organism size, and in particular growth curves, are often resource intensive because they involve many manual steps. Here we demonstrate a method for automated, high-throughput measurements of size and growth in individual aquatic invertebrates kept in microtiter well-plates. We use a spheroid counter (Cell3iMager, cc-5000) to automatically measure size of seven different freshwater invertebrate species. Further, we generated calibration curves (linear regressions, all p < 0.0001, r2 >=0.9 for Ceriodaphnoa dubia, Asellus aquaticus, Daphnia magna, Daphnia pulex; r2 >=0.8 for Hyalella azteca, Chironomus spec. larvae and Culex spec. larvae) to convert size measured on the spheroid counter to traditional, microscope based, length measurements, which follow the longest orientation of the body. Finally, we demonstrate semi-automated measurement of growth curves of individual daphnids (C. dubia and D. magna) over time and find that the quality of individual growth curves varies, partly due to methodological reasons. Nevertheless, this novel method could be adopted to other species and represents a step change in experimental throughput for measuring organisms’ shape, size and growth curves. It is also a significant qualitative improvement by enabling high-throughput assessment of interindividual variation of growth.

Publication - Biomedicines

Co Microencapsulation of Islets and MSC CellSaics Mosaic_Like Aggregates of MSCs and Recombinant Peptide Pieces and Therapeutic Effects of Their Subcutaneous Transplantation on Diabetes

The subcutaneous transplantation of microencapsulated islets has been extensively studied as a therapeutic approach for type I diabetes. However, due to the lower vascular density and strong inflammatory response in the subcutaneous area, there have been few reports of successfully normalized blood glucose levels. To address this issue, we developed mosaic-like aggregates comprised of mesenchymal stem cells (MSCs) and recombinant peptide pieces called MSC CellSaics, which provide a continuous release of angiogenic factors and anti-inflammatory cytokines. Our previous report revealed that the diabetes of immunodeficient diabetic model mice was reversed by the subcutaneous co-transplantation of the MSC CellSaics and rat islets. In this study, we focused on the development of immune-isolating microcapsules to co-encapsulate the MSC CellSaics and rat islets, and their therapeutic eciency via subcutaneous transplantation into immunocompetent diabetic model mice. As blood glucose level was monitored for 28 days following transplantation, the normalization rate of the new immuno-isolating microcapsules was confirmed to be significantly higher than those of the microcapsules without the MSC CellSaics, and the MSC CellSaics transplanted outside the microcapsules (p < 0.01). Furthermore, the number of islets required for the treatment was reduced. In the stained sections, a larger number/area of blood vessels was observed around the new immuno-isolating microcapsules, which suggests that angiogenic factors secreted by the MSC CellSaics through the microcapsules function locally for their enhanced efficacy.

Publication - PLOS ONE

Human Dynactin_Associated Protein Transforms NIH3T3 Cells to Generate Highly Vascularized Tumors with Weak Cell_Cell Interaction

Human dynactin-associated protein (dynAP) is a transmembrane protein that promotes Akt- Ser473 phosphorylation. Here, we report the oncogenic properties of dynAP. In contrast to control NIH3T3 cells expressing LacZ (NIH3T3LacZ), NIH3T3dynAP cells vigorously formed foci in two-dimensional culture, colonies on soft agar, and spheroids in anchoragedeficient three-dimensional culture. NIH3T3dynAP cells injected into nude mice produced tumors with abundant blood vessels and weak cell—cell contacts. Expression of dynAP elevated the level of rictor (an essential subunit of mTORC2) and promoted phosphorylation of FOXO3aSer253. FOXO3a is a transcriptional factor that stimulates expression of pro-apoptotic genes and phosphorylation of FOXO3a abrogates its function, resulting in promoted cell survival. Knockdown of rictor in NIH3T3dynAP cells reduced AktSer473 phosphorylation and formation of foci, colony in soft agar and spheroid, indicating that dynAP-induced activation of the mTORC2/AktSer473 pathway for cell survival contributes to cell transformation. E-cadherin and its mRNA were markedly reduced upon expression of dynAP, giving rise to cells with higher motility, which may be responsible for the weak cell-cell adhesion in tumors. Thus, dynAP could be a new oncoprotein and a target for cancer therapy.

Publication - BMC Cancer

Monospecific antibody targeting of CDH11 inhibits epithelial_to_mesenchymal transition and represses cancer stem cell_like phenotype by up_regulating miR_335 in metastatic breast cancer in v

Background: Metastasis is a leading cause of breast cancer mortality. The induction of epithelial-to-mesenchymal transition (EMT) and complex oncogenic signaling is a vital step in the evolution of highly metastatic and therapeutically-intractable breast cancer; necessitating novel target discovery or development of therapeutics that target metastatic breast cells (MBCs). Methods: To achieve this, this study employs a combination of in silico bioinformatics analyses, protein and transcript analyses, drug sensitivity assays, functional assays and animal studies. Results: The present study identified CDH11 as an inductor and/or facilitator of metastatic signaling, and biomarker of poor prognosis in MBCs. Furthermore, we showed that in the presence of CDH11-rich cancer-associated fibroblasts (CAFs), MCF7 and MDA-MB-231 MBC cell lines acquired enhanced metastatic phenotype with increased CDH11, β-catenin, vimentin, and fibronectin (FN) expression. We also demonstrated, for the first time to the best of our knowledge that exposure to anti- CDH11 antibody suppresses metastasis, reduces CDH11, FN and β-catenin expression, and abrogate the cancer stem cell (CSC)-like traits of MBC cells. Interestingly, ectopic expression of miR-335 suppressed CDH11, β-catenin and vimentin expression, in concert with attenuated metastatic and CSC potentials of the MBC cells; conversely, inhibition of miR-335 resulted in increased metastatic potential. Finally, corroborating the in silica and in vitro findings, in vivo assays showed that the administration of anti-CDH11 antibody or miR-335 mimic suppressed tumorigenesis and inhibited cancer metastasis. Conclusions: These findings validate our hypotheses that miR-335 mediates anti-CDH11 antibody therapy response and that an enhanced miR-335/CDH11 ratio elicits marked suppression of the MBC CSC-like and metastatic phenotypes, thus revealing a therapeutically-exploitable inverse correlation between CDH11-enhanced CSC-like and metastatic phenotype and miR-335 expression in MBCs. Thus, we highlight the therapeutic promise of humanized anti-CDH11 antibodies or miR-335- mimic, making a case for their clinical application as efficacious therapeutic option in patients with MBC.

Publication - PNAS

PAF promotes stemness and radioresistance of glioma

An integrated genomic and functional analysis to elucidate DNA damage signaling factors promoting self-renewal of glioma stem cells (GSCs) identified proliferating cell nuclear antigen (PCNA)- associated factor (PAF) up-regulation in glioblastoma. PAF is preferentially overexpressed in GSCs. Its depletion impairs maintenance of self-renewal without promoting differentiation and reduces tumorinitiating cell frequency. Combined transcriptomic and metabolomic analyses revealed that PAF supports GSC maintenance, in part, by influencing DNA replication and pyrimidine metabolism pathways. PAF interacts with PCNA and regulates PCNA-associated DNA translesion synthesis (TLS); consequently, PAF depletion in combination with radiation generated fewer tumorspheres compared with radiation alone. Correspondingly, pharmacological impairment of DNA replication and TLS phenocopied the effect of PAF depletion in compromising GSC self-renewal and radioresistance, providing preclinical proof of principle that combined TLS inhibition and radiation therapy may be a viable therapeutic option in the treatment of glioblastoma multiforme (GBM).

Publication - MDPI/Cancers

Preclinical Evaluation of the Novel Small_Molecule for Treating Drug_Resistant Colon Cancer via the LGR5Network and Reducing CAF Transformation

Colorectal cancer represents one of the most prevalent malignancies globally, with an estimated 140,000 new cases in the United States alone in 2019. Despite advancements in interventions, drug resistance occurs in virtually all patients diagnosed with late stages of colon cancer. Amplified epidermal growth factor receptor (EGFR) signaling is one of the most prevalent oncogenic drivers in patients and induces increased Janus kinase (JAK)/signal transduction and activator of transcription (STAT) and -catenin functions, all of which facilitate disease progression. Equally important, cancer-associated fibroblasts (CAFs) transformed by cancer cells within the tumor microenvironment (TME) further facilitate malignancy by secreting interleukin (IL)-6 and augmenting STAT3 signaling in colon cancer cells and promoting the generation of cancer stem-like cells (CSCs). Based on these premises, single-targeted therapeutics have proven ine ective for treating malignant colon cancer, and alternative multiple-targeting agents should be explored. Herein, we synthesized a tetracyclic heterocyclic azathioxanthone, MSI-N1014, and demonstrated its therapeutic potential both in vitro and in vivo. First, we used a co-culture system to demonstrate that colon cancer cells co-cultured with CAFs resulted in heightened 5-fluorouracil (5-FU) resistance and tumor sphere-forming ability and increased side populations, accompanied by elevated expression of cluster of di erentiation 44 (CD44), -catenin, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and ATP-binding cassette super-family G member 2 (ABCG2). MSI-N1014 suppressed cell viability, colony formation, and migration in both DLD1 and HCT116 cells. MSI-N1014 treatment led to decreased expressions of oncogenic markers, including mammalian target of rapamycin (mTOR), EGFR, and IL-6 and stemness markers such as CD44, -catenin, and LGR5. More importantly, MSI-N1014 treatment suppressed the transformation of CAFs, and was associated with decreased secretion of IL-6 and vascular endothelial growth factor (VEGF) by CAFs. Furthermore, MSI-N1014 treatment resulted in significantly reduced oncogenic properties, namely the migratory ability, tumor-sphere generation, and resistance against 5-FU. Notably, an increased level of the tumor suppressor, miR-142-3p, whose targets include LGR5, IL-6, and ABCG2, was detected in association with MSI-N1014 treatment. Finally, we demonstrated the therapeutic potential of MSI-N1014 in vivo, where combined treatment with MSI-N1014 and 5-FU led to the lowest tumor growth, followed by MSI-N1014 only, 5-FU, and the vehicle control. Tumor samples from the MSI-N1014 group showed markedly reduced expressions of LGR5, -catenin, IL-6, and mTOR, but increased expression of the tumor suppressor, miR-142-3p, according to qRT-PCR analysis. Collectively, we present preclinical support for the application of MSI-N1014 in treating 5-FU-resistant colon cancer cells. Further investigation is warranted to translate these findings into clinical settings.

Publication - Nature/Scientific Reports

Sertraline chlorprothixene and chlorpromazine characteristically interact with the REST binding site of the corepressor mSin3 showing medulloblastoma cell growth inhibitory activities

Dysregulation of repressor-element 1 silencing transcription factor REST/NRSF is related to several neuropathies, including medulloblastoma, glioblastoma, Huntington’s disease, and neuropathic pain. Inhibitors of the interaction between the N-terminal repressor domain of REST/NRSF and the PAH1 domain of its corepressor mSin3 may ameliorate such neuropathies. In-silico screening based on the complex structure of REST/NRSF and mSin3 PAH1 yielded 52 active compounds, including approved neuropathic drugs. We investigated their binding affinity to PAH1 by NMR, and their inhibitory activity toward medulloblastoma cell growth. Interestingly, three antidepressant and antipsychotic medicines, sertraline, chlorprothixene, and chlorpromazine, were found to strongly bind to PAH1. Multivariate analysis based on NMR chemical shift changes in PAH1 residues induced by ligand binding was used to identify compound characteristics associated with cell growth inhibition. Active compounds showed a new chemo-type for inhibitors of the REST/NRSF-mSin3 interaction, raising the possibility of new therapies for neuropathies caused by dysregulation of REST/NRSF.

Publication - Journal of Gastroentrology

Single cell analysis of Crohns disease patient_derived small intestinal organoids reveals disease activity dependent modification of stem cell properties

roles in the maintenance of homeostasis, and also in the regeneration of the damaged intestinal epithelia. However, whether the inflammatory environment of Crohn’s disease (CD) affects properties of resident small intestinal stem cells remain uncertain. Methods CD patient-derived small intestinal organoids were established from enteroscopic biopsy specimens taken from active lesions (aCD-SIO), or from mucosa under remission (rCD-SIO). Expression of ISC-marker genes in those organoids was examined by immunohistochemistry, and also by microfluid-based single-cell multiplex gene expression analysis. The ISC-specific function of organoid cells was evaluated using a single-cell organoid reformation assay. Results ISC-marker genes, OLFM4 and SLC12A2, were expressed by an increased number of small intestinal epithelial cells in the active lesion of CD. aCD-SIOs, rCDSIOs or those of non-IBD controls (NI-SIOs) were successfully established from 9 patients. Immunohistochemistry showed a comparable level of OLFM4 and SLC12A2 expression in all organoids. Single-cell gene expression data of 12 ISC-markers were acquired from a total of 1215 cells. t-distributed stochastic neighbor embedding analysis identified clusters of candidate ISCs, and also revealed a distinct expression pattern of SMOC2 and LGR5 in ISCcluster classified cells derived from aCD-SIOs. Single-cell organoid reformation assays showed significantly higher reformation efficiency by the cells of the aCD-SIOs compared with that of cells from NI-SIOs. Conclusions aCD-SIOs harbor ISCs with modified marker expression profiles, and also with high organoid reformation ability. Results suggest modification of small intestinal stem cell properties by unidentified factors in the inflammatory environment of CD.

Publication - BBRC

The anti inflammatory effect of the gut lactic acid bacteria-generated metabolite on monocytes

We evaluated the effect of gut bacterial metabolites of polyunsaturated fatty acids on inflammation and found that 10-oxo-cis-6,trans-11-octadecadienoic acid (gKetoC) strikingly suppressed LPS-induced IL-6 release from bone marrow-derived macrophages (BMMs), which was accompanied by reduced mRNA expression of Il6, TNF, and Il1b. gKetoC decreased the cAMP concentration in BMMs, suggesting that gKetoC stimulated G protein-coupled receptors. A Gq agonist significantly suppressed LPS-induced IL-6 expression in BMMs, whereas a Gi inhibitor partially abrogated gKetoC-mediated IL-6 suppression. Cytosolic Ca2þ was markedly increased by gKetoC, which was partly but not fully abrogated by an ion channel inhibitor. Taken together, these data suggest that gKetoC suppresses inflammatory cytokine expression in macrophages primarily through Gq and partially through Gi. gKetoC suppressed osteoclast development and IL-6 expression in synovial fibroblasts from rheumatoid arthritis (RA) patients, suggesting the beneficial effect of gKetoC on the prevention or treatment of RA.

Publication- Am Jour Can Res

Wild type TP53 defined gamma_secretase inhibitor sensitivity and synergistic activity with doxorubicin in GSCs

Glioblastoma (GBM) is the most common and lethal primary intracranial tumor. Aggressive surgical resection plus radiotherapy and temozolomide have prolonged patients’ median survival to only 14.6 months. Therefore, there is a critical need to develop novel therapeutic strategies for GBM. In this study, we evaluated the effect of NOTCH signaling intervention by gamma-secretase inhibitors (GSIs) on glioma sphere-forming cells (GSCs). GSI sensitivity exhibited remarkable selectivity among wild-type TP53 (wt-p53) GSCs. GSIs significantly impaired the sphere formation of GSCs harboring wt-p53. We also identified a concurrence between GSI sensitivity, NOTCH1 expression, and wt-p53 activity in GSCs. Through a series of gene editing and drug treatment experiments, we found that wt-p53 did not modulate NOTCH1 pathway, whereas NOTCH1 signaling positively regulated wt-p53 expression and activity in GSCs. Finally, GSIs (targeting NOTCH signaling) synergized with doxorubicin (activating wt-p53) to inhibit proliferation and induce apoptosis in wt-p53 GSCs. Taken together, we identified wt-p53 as a potential marker for GSI sensitivity in GSCs. Combining GSI with doxorubicin synergistically inhibited the proliferation and survival of GSCs harboring wt-p53.

Feature article- Gene Drug Discovery

Phenotypic Drug Discovery in 3D

Testing Drug Efficacy by Rapid Size Profiling Over Time with Tumor Microtissues

Deep Learning Literature

Poster

Label-free image analysis and its applications of human iPSC-derived intestinal organoids

In recent years, research on organoids in regenerative medicine and drug discovery research has developed rapidly. In particular, the research for producing intestinal organoids from induced pluripotent stem cells (iPS cells) and human biological tissues has been remarkably developed, and the culture conditions for each have been almost established. However, imaging and image analysis methods of organoids have not been established yet. Organoids are composed of heterogeneous cell populations, and in addition to exhibiting a non-uniform morphology for each organoid, cell populations other than the evaluation target are likely to be included in the image. For these reasons, only by using image processing with a simple parameter threshold, high-precision segmentation of organoids cannot be performed, and quantification by image analysis is difficult. In the case of image analysis using a fluorescent label, there is a possibility that the above problems can be overcome, but today, the importance of label-free analysis without using a fluorescent label is increasing. In general, a three-dimensional culture method with an extracellular matrix is used for organoid culture, which makes imaging itself and image analysis more difficult. Therefore, in this study, we attempted a bright field image analysis of human iPSC-derived intestinal organoid using deep learning technology, which utilized in various fields. Since individual organoids cultured in Matrigel are located at different heights, all-in-focus images were obtained by Z-stack imaging. By using deep learning on the acquired image, only organoids exhibiting specific morphologies were segmented from the image, and feature quantities such as organoid count, diameter and area were calculated. Cell3iMager duos (SCREEN Holdings Co., Ltd.) was used for this imaging and image analysis. In this presentation, we will discuss the label-free analysis of organoids and their applications.

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