Kazuya Arai

A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516

Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. The half maximal inhibitory concentration (IC50) of SB-525334 and SU9516 were 0.31 μM and 1.21 μM, respectively, while IC50 of SB431542 was 2.38 μM. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.

Katanin p60 contributes to microtubule instability around the midbody and facilitates cytokinesis in rat cells.

The completion of cytokinesis is crucial for mitotic cell division. Cleavage furrow ingression is followed by the breaking and resealing of the intercellular bridge, but the detailed mechanism underlying this phenomenon remains unknown. Katanin is a microtubule-severing protein comprised of an AAA ATPase subunit and an accessory subunit designated as p60 and p80, respectively. Localization of katanin p60 was observed at the midzone to midbody from anaphase to cytokinesis in rat cells, and showed a ring-shaped distribution in the gap between the inside of the contractile ring and the central spindle bundle in telophase. Katanin p60 did not bind with p80 at the midzone or midbody, and localization was shown to be dependent on microtubules. At the central spindle and the midbody, no microtubule growth plus termini were seen with katanin p60, and microtubule density was inversely correlated with katanin p60 density in the region of katanin p60 localization that seemed to lead to microtubule destabilization at the midbody. Inhibition of katanin p60 resulted in incomplete cytokinesis by regression and thus caused the appearance of binucleate cells. These results suggest that katanin p60 contributes to microtubule instability at the midzone and midbody and facilitates cytokinesis in rat cells.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

Abstract 610: Effect of scaffold and growth factors on anti-cancer drug screening with multicellular spheroids: mimicking in vivo response

Background: For in vitro cancer research, cancer microenvironment is being increasingly recognized as a key factor. In the past, several 3D cell culture models have been tested as the powerful method for reproducing cancer microenvironment. However, in some cases, the growth and drug sensitivity of cells grown on anchorage-independent 3D culture models have been different to that of cells grown in vivo. Further, development of the 3D culture model including the physical (the cell-ECM interaction (scaffold)) and chemical factor (cytokine) are essential for mimicking cancer microenvironment in vitro. Therefore, we explored the effect of the growth factor dependent proliferation and drug sensitivity in scaffold/scaffold-free culture models, and try to understand which method is mimicking in vivo response due to stimulation and suitable for anti-cancer drug development. Methods: We cultured lung cancer, breast cancer, pancreatic cancer and ovarian cancer cell line on monolayer, Matrigel (ECM-embedded 3D culture, scaffold type), NanoCuluture Plate (NCP, a gel-free scaffold type 3D culture plate) and Low adhesion round-bottom plate (scaffold-free 3D culture plate). For growth curve experiment upon addition of growth factors (EGF, HB-EGF, FGF, HRG), we evaluated cell viabilities by ATP assay in several cancer cell lines. We examined drug sensitivities of cancer cell lines against clinically active anti-cancer agents by addition of growth factors. Results: Growth factor promoted the proliferation of cancer cell lines cultured on NCP while it was not enhanced the cell growth when cultured on 2D, Matrigel and Low adhesion round-bottom plate. In drug response, the cells grown on NCP by growth factor stimulation was more sensitive to drug than 2D and other 3D culture models. Conclusion: Differences of the cell growth and drug sensitivity were appeared by effect of scaffold and growth factor. Growth factor dependent cell proliferation and drug sensitivity on NCP were similar to in vivo behavior (1). These results demonstrated that scaffold and growth factor are the novel approach to be mimicking the cancer microenvironment in vitro. Therefore, it is obvious that NCP can be used as a suitable 3D culture model for mimicking in vivo response and anti-cancer drug development. 1. J Cell Sci. 2009 Dec 1;122(Pt 23):4277-86. Citation Format: Norio Masuda, M. Mamunur Rahman, Kazuya Arai, Atsushi Mizuno, Manabu Itoh. Effect of scaffold and growth factors on anti-cancer drug screening with multicellular spheroids: mimicking in vivo response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 610.

Abstract 303: Effect of scaffold on drug sensitivity of multicellular spheroids: Which method is close to in vivo and suitable for HTS

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Three-dimensional (3D) cell culture models are more extensive to be better models than two-dimensional (2D) cell culture models due to enriched cellular signaling pathways, cell to-cell contact, more representative of tissue morphology, and predictive of drug responses in vivo. Different drugs screening and various biological responses between 2D and 3D cell culture models have been reported. However, very little information is available on cell function and/or drug susceptibility caused by a difference in methodology of three dimensional culture. Here, we compared drug responses of various cancer cells against distinctive anti-cancer drugs when grown in monolayer, scaffold 3D culture (Matrigel, NanoCuluture Plate (NCP)) or scaffold-free 3D culture models (ultra-low attachment round bottom plate), and also confirmed which method is suitable for high-throughput screening for robust three-dimensional screening model. Method: We examined drug sensitivities of different lung cancer (A549, H226, H1650), breast cancer (SKBR-3, JIMT1), and prostate cancer (DU145) cell lines under monolayer or 3D cell culture conditions against several anti-cancer agents i.e. molecular targeted agent (Gefitinib, Afatinib, Temsirolimus, Vemurafenib, Vismodegib, Sorafenib), demethylation agent (Azacytidine, Decitabine), anti-microtubule agent (Docetaxel), alkylating agent (Cisplatin, Irinotecan), antimetabolite agent (5-FU, Gemcitabine, Methotrexate). The conditions of these assays are optimized for each culture method. Cell proliferation was measured by ATP assay. Furthermore, we analyzed the biological/ morphological differences among 3D culture methods (scaffold or scaffold-free culture models). Result: Large variations in drug responses were observed among the different cell culture models. Cells grown in 3D scaffold culture model (NCP) were more drug sensitive than other cell culture models. Especially docetaxel, 5-FU, and methotrexate showed clear growth inhibition in three dimensional scaffold culture models (NCP) than scaffold-free culture models (round-bottom). Conclusion: In this study, we showed that scaffold 3D culture is appropriate for anti-cancer drug-susceptibility test. We also proposed that NanoCuluture plate which is 3D scaffold culture model, can be used as a sophisticated high-throughput three-dimensional drug screening model. Citation Format: Norio Masuda, Atsushi Mizuno, M.Mamunur Rahman, Kazuya Arai, Manabu Itoh. Effect of scaffold on drug sensitivity of multicellular spheroids: Which method is close to in vivo and suitable for HTS. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 303. doi:10.1158/1538-7445.AM2015-303

Abstract 697: Development of spheroid based high-throughput screening of cell-cell adhesion inhibitors to reverse acquired multicellular resistance

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Solid tumors are more resistant to chemo- and radio-therapy than in vitro cancer cells under conventional monolayer culture condition. This phenomenon can be explained by “multicellular resistance” (MCR). It is considered that one of approach for keeping out cancer cells from acquired MCR is inhibition of cell-cell/cell-matrix adherence. Several therapeutic approaches increasing effect of chemo- and radio-therapy by preventing MCR are proposed. However, reports of the approaches are still few. Therefore, we tried to develop a spheroid-based high-throughput screening method of cell-cell adherence inhibitor, for discovery of drug capable of reversing the acquired MCR. Method: For spheroid formation, A549 cells were cultured under three-dimensional condition using NanoCulture® Plate, which is compatible with imaging and HTS systems. After 3 days culture, A549 spheroids were treated with library compounds for 4 days. To evaluate a degree of cell-cell adherence of the spheroids, we used Hypoxia Probe, which is a phosphorescence substrate for visualizing of intra-spheroid hypoxicity. And the signals were detected using Celigo® imaging cytometer. Results: In NanoCulture® Plate, A549 cells formed a number of spheroids. Intra-spheroid hypoxicity was correlated with degree of cell-cell adherence. After screening of inhibitor of cell-cell adherence without cytotoxity, several candidates were evaluated the ability as chemosensitizing agent. Combination treatment with the hit compound and anticancer drug was more effective than each drug alone. Conclusion: We developed a novel high throughput screening method of cell-cell adherence inhibitors. And we acquired several hit compounds capable of reversing the acquired MCR. Note: This abstract was not presented at the meeting. Citation Format: Kazuya Arai, Manabu Itho, Atsushi Mizuno, Hiromi Miura. Development of spheroid based high-throughput screening of cell-cell adhesion inhibitors to reverse acquired multicellular resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 697. doi:10.1158/1538-7445.AM2014-697

Abstract 5525: Development of novel high-throughput screening assay system for exploring EMT inhibitors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: EMT (Epitherial-Mesenchymal transition) is a significant event in tumor metastasis and malignancy. Cancer cells are stimulated by EMT-inducible agonists from the surrounding stromal cells, and cause transformation such as weakening of cell-cell adhesion and the accompanying acceleration of cell migration and invasion. Inhibition of EMT is considered to enable controlling of malignant transformation. In this study, we developed a high-throughput assay system of EMT inhibitor screen in 3D cell culture. Method: We adopted 3D cell culture method on NanoCulture® Plate (NCP), a scaffold type spheroid culture plate, as a culture method which can confirm spheroid morphologies. TGF-β and its inhibitor, SB431542, were used as an EMT inducer and as a positive control for EMT inhibitor, respectively. Expressions of E-cadherin, N-cadherin, vimentin and zeb-1, represent EMT maker genes, were evaluated by qRT-PCR. As hypoxia inside the spheroids reflected the spheroid size and cell-cell adhesion strength, we visualized the intra-spheroid hypoxia, and used it as an indicator for EMT induction and inhibition. Result: A549 cell spheroids gradually collapsed, by the weakening of cell-cell adhesion, and cells migrating out from the spheroids, by treating with TGF-β. This collapse of the spheroids was inhibited by SB431542 treatment. SB431542 treatment also accompanied inhibition of E-cadherin down-regulation, and N-cadherin, vimentin and zeb-1 up-regulation. These results indicate that this method is clearly reproducing EMT and its inhibition. And we elucidated that intra-spheroid hypoxicity was closely correlated with spheroid morphology. From this, it is demonstrated that this method is simple but high-precision assay. Conclusion: We developed a high-throughput assay system for EMT inhibitor screen, by evaluating intra-spheroid hypoxicity as an indicator for alternation of the spheroid morphology. This method is an unique method which enables to evaluate EMT condition, by monitoring the collapse of spheroids directly. We are now running a screen for EMT inhibitor candidate compounds, by using this assay system. Citation Format: Kazuya Arai, Ruriko Sakamoto, Manabu Itoh, Hiromi Miura, Manami Shimomura, Tetsuya Nakatsura. Development of novel high-throughput screening assay system for exploring EMT inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5525. doi:10.1158/1538-7445.AM2013-5525

Abstract LB-502: Development of three dimensional cancer cell-CAF co-culture system

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose A large number of cell lines have been established and used for cancer research. Monolayer culture of cancer cells alone is a simplified and effective method, however, it does not always reflect the physiological conditions. In this study, we tried to establish a simple co-culture method of cancer cells and CAF (cancer associated fibroblast), to develop three dimensional model of stroma rich tumor with cells such as pancreatic cancer and scirrhous cancer. Method Human tumor tissue samples were obtained from patients who had undergone surgery at the National Cancer Center Hospital East, Japan. Written informed consents were obtained from all patients. BxPC-3 (pancreatic cancer cell line) and A549 (lung cancer cell line) were stained with red-fluorescent dye, PKH-26. Pancreatic cancer derived- and lung cancer derived-CAF were stained with green-fluorescent dye, PKH-67. Cancer cells and CAF are seeded into the microtiter plates for 3D cell culture (NanoCulture® Plate) at a same cell number, and spheroid formation was measured by a fluorescent time lapse microscopy for 3-days. Result BxPC-3 (red) and Pancreatic CAF (green) merged well and formed mosaic spheroids. BxPC-3 (red) and lung CAF (green) did not merge and separately formed spheroids of each cell types. A549 and lung CAF merged, but A549 and Pancreatic CAF did not merge clearly. It is suggested that a specific recognition mechanisms exist by organ types or tumor types, with the adhesion between cancer cell and CAF. A) BxPC-3 and Pancreatic CAF B) BxPC-3 and lung CAF Conclusion In this study, mosaic spheroid were formed by co-culturing of same organ derived cancer cells and CAF. This co-culture system is expected as an effective tool for cancer microenvironment study and/or drug screening targeting with stroma rich tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-502. doi:1538-7445.AM2012-LB-502