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.

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.

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

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.

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).