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Dilloo S, Whittaker A, Chang X, D’Amen E, Spisni E, Hrelia S, Angeloni C, Malaguti M, Dinelli G, Truzzi F. Administration of Spermidine and Eugenol Demonstrates Anti-Tumorigenic Efficacy on Metastatic SW620 and Primary Caco-2 Colorectal Cancer Spheroids. Int J Mol Sci 2024; 25:13362. [PMID: 39769127 PMCID: PMC11679521 DOI: 10.3390/ijms252413362] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2024] [Revised: 12/06/2024] [Accepted: 12/09/2024] [Indexed: 01/11/2025] Open
Abstract
The anti-cancer potential of eugenol (EUG) is well recognized, whereas that of spermidine (SPD) is subject to dispute and requires further research. The anti-tumorigenic potential of wheat germ SPD (150 µM) and clove EUG (100 µM), alone, in combination as SPD+EUG (50 µM + 100 µM) and, as a supplement (SUPPL; 0.6 µM SPD + 50 µM EUG), was investigated on both metastatic SW620 and primary Caco-2 colorectal cancer (CRC) spheroids. Compared to untreated controls, all treatments significantly reduced the vitality and spheroid area, increased the necrotic area, and induced apoptosis on both cell-type spheroids after 96 h, with a reduced migration evident in 2D (two-dimensional) cultures after 48 h. The comparable anti-CRC effects of the SPD+EUG and the SUPPL reflected a wide-range dose efficacy of SPD and EUG. It is of note that SPD+EUG induced a synergistic effect on the increased caspase-3 expression and reduced the migration percentage in SW620. In more physiologically relevant intestinal equivalents (healthy enterocytes [NCM460], fibroblasts [L929], and monocytes [U937]) containing embedded SW620/Caco-2 spheroids, SPD+EUG administration significantly reduced the spheroid CEA marker and proliferation, whilst simultaneously increasing occludin, autophagy LC3-II expression, and monocyte differentiation, compared to the control models. Exogenous SPD, alone and in combination with EUG, displayed an anti-CRC potential on tumor growth and metastasis, and warrants further investigation.
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Affiliation(s)
- Silvia Dilloo
- Department of Agricultural and Food Sciences, Alma Mater Studiorum—University of Bologna, 40127 Bologna, Italy; (S.D.); (A.W.); (X.C.); (E.D.); (G.D.)
| | - Anne Whittaker
- Department of Agricultural and Food Sciences, Alma Mater Studiorum—University of Bologna, 40127 Bologna, Italy; (S.D.); (A.W.); (X.C.); (E.D.); (G.D.)
| | - Xinyue Chang
- Department of Agricultural and Food Sciences, Alma Mater Studiorum—University of Bologna, 40127 Bologna, Italy; (S.D.); (A.W.); (X.C.); (E.D.); (G.D.)
| | - Eros D’Amen
- Department of Agricultural and Food Sciences, Alma Mater Studiorum—University of Bologna, 40127 Bologna, Italy; (S.D.); (A.W.); (X.C.); (E.D.); (G.D.)
| | - Enzo Spisni
- Department of Biological, Geological, and Environmental Sciences, Alma Mater Studiorum—University of Bologna, 40127 Bologna, Italy;
| | - Silvana Hrelia
- Department for Life Quality Studies, Alma Mater Studiorum—University of Bologna, 47921 Rimini, Italy; (S.H.); (C.A.); (M.M.)
| | - Cristina Angeloni
- Department for Life Quality Studies, Alma Mater Studiorum—University of Bologna, 47921 Rimini, Italy; (S.H.); (C.A.); (M.M.)
| | - Marco Malaguti
- Department for Life Quality Studies, Alma Mater Studiorum—University of Bologna, 47921 Rimini, Italy; (S.H.); (C.A.); (M.M.)
| | - Giovanni Dinelli
- Department of Agricultural and Food Sciences, Alma Mater Studiorum—University of Bologna, 40127 Bologna, Italy; (S.D.); (A.W.); (X.C.); (E.D.); (G.D.)
| | - Francesca Truzzi
- Department of Agricultural and Food Sciences, Alma Mater Studiorum—University of Bologna, 40127 Bologna, Italy; (S.D.); (A.W.); (X.C.); (E.D.); (G.D.)
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Cheng YW, Hsieh YC, Sun YS, Wang YH, Yang YW, Lo KY. Using microfluidic and conventional platforms to evaluate the effects of lanthanides on spheroid formation. Toxicology 2024; 508:153931. [PMID: 39222830 DOI: 10.1016/j.tox.2024.153931] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2024] [Revised: 08/16/2024] [Accepted: 08/17/2024] [Indexed: 09/04/2024]
Abstract
Metastasis contributes to the increased mortality rate of cancer, but the intricate mechanisms remain unclear. Cancer cells from a primary tumor invade nearby tissues and access the lymphatic or circulatory system. If these cells manage to survive and extravasate from the vasculature into distant tissues and ultimately adapt to survive, they will proliferate and facilitate malignant tumor formation. Traditional two-dimensional (2D) cell cultures offer a rapid and convenient method for validating the efficacy of anticancer drugs within a reasonable cost range, but their utility is limited because of tumors' high heterogeneity in vivo and spatial complexities. Three-dimensional (3D) cell cultures that mimic the physiological conditions of cancer cells in vivo have gained considerable interest. In these cultures, cells assemble into spheroids through gravity, magnetic forces, or their low-adhesion to the plates. Although these approaches address some of the limitations of 2D cultures, they often require a considerable amount of time and cost. Therefore, this study aims to enhance the effectiveness of 3D culture techniques by using microfluidic systems to provide a high-throughput and sensitive pipeline for drug screening. Using these systems, we studied the effects of lanthanide elements, which have garnered interest in cancer treatment, on spheroid formation and cell spreading. Our findings suggest that these elements alter the compactness of cell spheroids and decrease cell mobility.
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Affiliation(s)
- Yu-Wen Cheng
- Department of Agricultural Chemistry, National Taiwan University Taipei City 10617, Taiwan
| | - Yu-Chen Hsieh
- Department of Agricultural Chemistry, National Taiwan University Taipei City 10617, Taiwan
| | - Yung-Shin Sun
- Department of Physics, Fu-Jen Catholic University, New Taipei City 24205, Taiwan
| | - Yu-Hsun Wang
- Department of Physics, Fu-Jen Catholic University, New Taipei City 24205, Taiwan
| | - Ya-Wen Yang
- Department of Surgery, National Taiwan University Hospital, Taipei City 100225, Taiwan.
| | - Kai-Yin Lo
- Department of Agricultural Chemistry, National Taiwan University Taipei City 10617, Taiwan.
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Tudor M, Popescu RC, Irimescu IN, Rzyanina A, Tarba N, Dinischiotu A, Craciun L, Esanu TR, Vasile E, Hotnog AT, Radu M, Mytsin G, Mihailescu M, Savu DI. Enhancing Proton Radiosensitivity of Chondrosarcoma Using Nanoparticle-Based Drug Delivery Approaches: A Comparative Study of High- and Low-Energy Protons. Int J Mol Sci 2024; 25:11481. [PMID: 39519034 PMCID: PMC11546389 DOI: 10.3390/ijms252111481] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2024] [Revised: 10/20/2024] [Accepted: 10/23/2024] [Indexed: 11/16/2024] Open
Abstract
To overcome chondrosarcoma's (CHS) high chemo- and radioresistance, we used polyethylene glycol-encapsulated iron oxide nanoparticles (IONPs) for the controlled delivery of the chemotherapeutic doxorubicin (IONPDOX) to amplify the cytotoxicity of proton radiation therapy. Human 2D CHS SW1353 cells were treated with protons (linear energy transfer (LET): 1.6 and 12.6 keV/µm) with and without IONPDOX. Cell survival was assayed using a clonogenic test, and genotoxicity was tested through the formation of micronuclei (MN) and γH2AX foci, respectively. Morphology together with spectral fingerprints of nuclei were measured using enhanced dark-field microscopy (EDFM) assembled with a hyperspectral imaging (HI) module and an axial scanning fluorescence module, as well as scanning electron microscopy (SEM) coupled with energy-dispersive X-Ray spectroscopy (EDX). Cell survival was also determined in 3D SW3153 spheroids following treatment with low-LET protons with/without the IONPDOX compound. IONPDOX increased radiosensitivity following proton irradiation at both LETs in correlation with DNA damage expressed as MN or γH2AX. The IONPDOX-low-LET proton combination caused a more lethal effect compared to IONPDOX-high-LET protons. CHS cell biological alterations were reflected by the modifications in the hyperspectral images and spectral profiles, emphasizing new possible spectroscopic markers of cancer therapy effects. Our findings show that the proposed treatment combination has the potential to improve the management of CHS.
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Affiliation(s)
- Mihaela Tudor
- Department of Life and Environmental Physics, Horia Hulubei National Institute of Physics and Nuclear Engineering, Reactorului 30, P.O. Box MG-6, 077125 Magurele, Romania; (M.T.); (R.C.P.); (M.R.)
- Faculty of Biology, University of Bucharest, Splaiul Independentei 91-95, 050095 Bucharest, Romania;
| | - Roxana Cristina Popescu
- Department of Life and Environmental Physics, Horia Hulubei National Institute of Physics and Nuclear Engineering, Reactorului 30, P.O. Box MG-6, 077125 Magurele, Romania; (M.T.); (R.C.P.); (M.R.)
- Department of Bioengineering and Biotechnology, Faculty of Medical Engineering, National University for Science and Technology Politehnica of Bucharest, Gheorghe Polizu Street, 1-7, 011061 Bucharest, Romania
| | - Ionela N. Irimescu
- Applied Sciences Doctoral School, National University for Science and Technology Politehnica of Bucharest, 060042 Bucharest, Romania;
| | - Ann Rzyanina
- Laboratory of Nuclear Problems, Joint Institute for Nuclear Research, 6 Joliot-Curie Street, 141980 Dubna, Moscow Region, Russia; (A.R.); (G.M.)
| | - Nicolae Tarba
- Doctoral School of Computer Sciences, National University for Science and Technology Politehnica of Bucharest, 060042 Bucharest, Romania;
| | - Anca Dinischiotu
- Faculty of Biology, University of Bucharest, Splaiul Independentei 91-95, 050095 Bucharest, Romania;
| | - Liviu Craciun
- Radiopharmaceutical Research Centre, Horia Hulubei National Institute of Physics and Nuclear Engineering, 077125 Magurele, Romania; (L.C.); (T.R.E.)
| | - Tiberiu Relu Esanu
- Radiopharmaceutical Research Centre, Horia Hulubei National Institute of Physics and Nuclear Engineering, 077125 Magurele, Romania; (L.C.); (T.R.E.)
| | - Eugeniu Vasile
- Faculty of Applied Physics, National University for Science and Technology Politehnica of Bucharest, 060042 Bucharest, Romania;
| | - Andrei Theodor Hotnog
- Applied Nuclear Physics Department, Horia Hulubei National Institute of Physics and Nuclear Engineering, Reactorului 30, P.O. Box MG-6, 077125 Magurele, Romania;
| | - Mihai Radu
- Department of Life and Environmental Physics, Horia Hulubei National Institute of Physics and Nuclear Engineering, Reactorului 30, P.O. Box MG-6, 077125 Magurele, Romania; (M.T.); (R.C.P.); (M.R.)
| | - Gennady Mytsin
- Laboratory of Nuclear Problems, Joint Institute for Nuclear Research, 6 Joliot-Curie Street, 141980 Dubna, Moscow Region, Russia; (A.R.); (G.M.)
| | - Mona Mihailescu
- Holographic Imaging and Processing Laboratory, Physics Department, National University for Science and Technology Politehnica of Bucharest, 060042 Bucharest, Romania;
- Centre for Research in Fundamental Sciences Applied in Engineering, National University for Science and Technology Politehnica of Bucharest, 060042 Bucharest, Romania
| | - Diana Iulia Savu
- Department of Life and Environmental Physics, Horia Hulubei National Institute of Physics and Nuclear Engineering, Reactorului 30, P.O. Box MG-6, 077125 Magurele, Romania; (M.T.); (R.C.P.); (M.R.)
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Kang Y, Na J, Karima G, Amirthalingam S, Hwang NS, Kim HD. Mesenchymal Stem Cell Spheroids: A Promising Tool for Vascularized Tissue Regeneration. Tissue Eng Regen Med 2024; 21:673-693. [PMID: 38578424 PMCID: PMC11187036 DOI: 10.1007/s13770-024-00636-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Revised: 02/29/2024] [Accepted: 03/05/2024] [Indexed: 04/06/2024] Open
Abstract
BACKGROUND Mesenchymal stem cells (MSCs) are undifferentiated cells that can differentiate into specific cell lineages when exposed to the right conditions. The ability of MSCs to differentiate into particular cells is considered very important in biological research and clinical applications. MSC spheroids are clusters of MSCs cultured in three dimensions, which play an important role in enhancing the proliferation and differentiation of MSCs. MSCs can also participate in vascular formation by differentiating into endothelial cells and secreting paracrine factors. Vascularization ability is essential in impaired tissue repair and function recovery. Therefore, the vascularization ability of MSCs, which enhances angiogenesis and accelerates tissue healing has made MSCs a promising tool for tissue regeneration. However, MSC spheroids are a relatively new research field, and more research is needed to understand their full potential. METHODS In this review, we highlight the importance of MSC spheroids' vascularization ability in tissue engineering and regenerative medicine while providing the current status of studies on the MSC spheroids' vascularization and suggesting potential future research directions for MSC spheroids. RESULTS Studies both in vivo and in vitro have demonstrated MSC spheroids' capacity to develop into endothelial cells and stimulate vasculogenesis. CONCLUSION MSC spheroids show potential to enhance vascularization ability in tissue regeneration. Yet, further research is required to comprehensively understand the relationship between MSC spheroids and vascularization mechanisms.
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Affiliation(s)
- Yoonjoo Kang
- Department of IT Convergence (Brain Korea Plus 21), Korea National University of Transportation, Chungju, 27469, Republic of Korea
| | - Jinwoo Na
- Department of Polymer Science and Engineering, Korea National University of Transportation, 50 Daehak-ro, Chungju, 27469, Republic of Korea
| | - Gul Karima
- Department of Polymer Science and Engineering, Korea National University of Transportation, 50 Daehak-ro, Chungju, 27469, Republic of Korea
| | - Sivashanmugam Amirthalingam
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, 08826, Republic of Korea
- Institute of Engineering Research, Seoul National University, Seoul, 08826, Republic of Korea
| | - Nathaniel S Hwang
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, 08826, Republic of Korea
- Institute of Engineering Research, Seoul National University, Seoul, 08826, Republic of Korea
| | - Hwan D Kim
- Department of IT Convergence (Brain Korea Plus 21), Korea National University of Transportation, Chungju, 27469, Republic of Korea.
- Department of Polymer Science and Engineering, Korea National University of Transportation, 50 Daehak-ro, Chungju, 27469, Republic of Korea.
- Department of Biomedical Engineering, Korea National University of Transportation, Chungju, 27469, Republic of Korea.
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Goyeneche AA, Lasiste JME, Abdouh M, Bustamante P, Burnier JV, Burnier MN. Delineating three-dimensional behavior of uveal melanoma cells under anchorage independent or dependent conditions. Cancer Cell Int 2024; 24:180. [PMID: 38783299 PMCID: PMC11118898 DOI: 10.1186/s12935-024-03350-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2023] [Accepted: 04/29/2024] [Indexed: 05/25/2024] Open
Abstract
BACKGROUND Although rare, uveal melanoma (UM) is a life-threatening malignancy. Understanding its biology is necessary to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools that incorporate physical and spatial cues that better mimic tumor biology and in turn deliver more predictive preclinical data. Herein, we comprehensively characterize UM cells under different 3D culture settings as a suitable model to study tumor cell behavior and therapeutic intervention. METHODS Six UM cell lines were tested in two-dimensional (2D) and 3D-culture conditions. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, and on hanging drops, with or without methylcellulose. Cultures were analyzed for multicellular tumor structures (MCTs) development by phase contrast and confocal imaging, and cell wellbeing was assessed based on viability, membrane integrity, vitality, apoptotic features, and DNA synthesis. Vascular endothelial growth factor (VEGF) production was evaluated under hypoxic conditions for cell function analysis. RESULTS UM cells cultured following anchorage-free methods developed MCTs shaped as spheres. Regardless of their sizes and degree of compaction, these spheres displayed an outer ring of viable and proliferating cells, and a core with less proliferating and apoptotic cells. In contrast, UM cells maintained under anchorage-dependent conditions established several morphological adaptations. Some remained isolated and rounded, formed multi-size irregular aggregates, or adopted a 2D-like flat appearance. These cells invariably conserved their metabolic activity and conserved melanocytic markers (i.e., expression of Melan A/Mart-1 and HMB45). Notably, under hypoxia, cells maintained under 3D conditions secrete more VEGF compared to cells cultured under 2D conditions. CONCLUSIONS Under an anchorage-free environment, UM cells form sphere-like MCTs that acquire attributes reminiscent of abnormal vascularized solid tumors. UM cells behavior in anchorage-dependent manner exposed diverse cells populations in response to cues from an enriched extracellular matrix proteins (ECM) environment, highlighting the plasticity of UM cells. This study provides a 3D cell culture platform that is more predictive of the biology of UM. The integration of such platforms to explore mechanisms of ECM-mediated tumor resistance, metastatic abilities, and to test novel therapeutics (i.e., anti-angiogenics and immunomodulators) would benefit UM care.
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Affiliation(s)
- Alicia A Goyeneche
- The MUHC-McGill University Ocular Pathology & Translational Research Laboratory, Research Institute of the McGill University Health Centre, Montreal, Canada.
- Cancer Research Program, Research Institute of the McGill University Health Centre, Montreal, Canada.
- Experimental Pathology Unit, Department of Pathology, McGill University, Montreal, Canada.
| | - Jade M E Lasiste
- The MUHC-McGill University Ocular Pathology & Translational Research Laboratory, Research Institute of the McGill University Health Centre, Montreal, Canada
| | - Mohamed Abdouh
- The MUHC-McGill University Ocular Pathology & Translational Research Laboratory, Research Institute of the McGill University Health Centre, Montreal, Canada
- Cancer Research Program, Research Institute of the McGill University Health Centre, Montreal, Canada
| | - Prisca Bustamante
- The MUHC-McGill University Ocular Pathology & Translational Research Laboratory, Research Institute of the McGill University Health Centre, Montreal, Canada
| | - Julia V Burnier
- The MUHC-McGill University Ocular Pathology & Translational Research Laboratory, Research Institute of the McGill University Health Centre, Montreal, Canada
- Cancer Research Program, Research Institute of the McGill University Health Centre, Montreal, Canada
- Experimental Pathology Unit, Department of Pathology, McGill University, Montreal, Canada
- Department of Oncology, McGill University, Montreal, Canada
| | - Miguel N Burnier
- The MUHC-McGill University Ocular Pathology & Translational Research Laboratory, Research Institute of the McGill University Health Centre, Montreal, Canada
- Cancer Research Program, Research Institute of the McGill University Health Centre, Montreal, Canada
- Experimental Pathology Unit, Department of Pathology, McGill University, Montreal, Canada
- Department of Oncology, McGill University, Montreal, Canada
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Biju TS, Priya VV, Francis AP. Role of three-dimensional cell culture in therapeutics and diagnostics: an updated review. Drug Deliv Transl Res 2023; 13:2239-2253. [PMID: 36971997 PMCID: PMC10042111 DOI: 10.1007/s13346-023-01327-6] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/06/2023] [Indexed: 03/29/2023]
Abstract
Drug development and testing are a tedious and expensive process with a high degree of uncertainty in the clinical success and preclinical validation of manufactured therapeutic agents. Currently, to understand the drug action, disease mechanism, and drug testing, most therapeutic drug manufacturers use 2D cell culture models to validate the drug action. However, there are many uncertainties and limitations with the conventional use of 2D (monolayer) cell culture models for drug testing that are primarily attributed due to poor mimicking of cellular mechanisms, disturbance in environmental interaction, and changes in structural morphology. To overcome such odds and difficulties in the preclinical validation of therapeutic medications, newer in vivo drug testing cell culture models with higher screening efficiencies are required. One such promising and advanced cell culture model reported recently is the "three-dimensional cell culture model." The 3D cell culture models are reported to show evident benefits over conventional 2D cell models. This review article outlines and describes the current advancement in cell culture models, their types, significance in high-throughput screening, limitations, applications in drug toxicity screening, and preclinical testing methodologies to predict in vivo efficacy.
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Affiliation(s)
- Tina Sara Biju
- Centre of Molecular Medicine and Diagnostics (COMManD), Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, 600077, India
| | - Veeraraghavan Vishnu Priya
- Centre of Molecular Medicine and Diagnostics (COMManD), Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, 600077, India
| | - Arul Prakash Francis
- Centre of Molecular Medicine and Diagnostics (COMManD), Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, 600077, India.
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Carannante V, Wiklund M, Önfelt B. In vitro models to study natural killer cell dynamics in the tumor microenvironment. Front Immunol 2023; 14:1135148. [PMID: 37457703 PMCID: PMC10338882 DOI: 10.3389/fimmu.2023.1135148] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Accepted: 06/05/2023] [Indexed: 07/18/2023] Open
Abstract
Immunotherapy is revolutionizing cancer therapy. The rapid development of new immunotherapeutic strategies to treat solid tumors is posing new challenges for preclinical research, demanding novel in vitro methods to test treatments. Such methods should meet specific requirements, such as enabling the evaluation of immune cell responses like cytotoxicity or cytokine release, and infiltration into the tumor microenvironment using cancer models representative of the original disease. They should allow high-throughput and high-content analysis, to evaluate the efficacy of treatments and understand immune-evasion processes to facilitate development of new therapeutic targets. Ideally, they should be suitable for personalized immunotherapy testing, providing information for patient stratification. Consequently, the application of in vitro 3-dimensional (3D) cell culture models, such as tumor spheroids and organoids, is rapidly expanding in the immunotherapeutic field, coupled with the development of novel imaging-based techniques and -omic analysis. In this paper, we review the recent advances in the development of in vitro 3D platforms applied to natural killer (NK) cell-based cancer immunotherapy studies, highlighting the benefits and limitations of the current methods, and discuss new concepts and future directions of the field.
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Affiliation(s)
- Valentina Carannante
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden
| | - Martin Wiklund
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden
| | - Björn Önfelt
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden
- Center for Infectious Medicine, Department of Medicine Huddinge, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden
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Roberge CL, Miceli RT, Murphy LR, Kingsley DM, Gross RA, Corr DT. Sophorolipid Candidates Demonstrate Cytotoxic Efficacy against 2D and 3D Breast Cancer Models. JOURNAL OF NATURAL PRODUCTS 2023; 86:1159-1170. [PMID: 37104545 PMCID: PMC10760934 DOI: 10.1021/acs.jnatprod.2c00804] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
Sophorolipids are biosurfactants derived from the nonpathogenic yeasts such as Starmerella bombicola with potential efficacy in anticancer applications. Simple and cost-effective synthesis of these drugs makes them a promising alternative to traditional chemotherapeutics, pending their success in preliminary drug-screening. Drug-screening typically utilizes 2D cell monolayers due to their simplicity and ease of high-throughput assessment. However, 2D assays fail to capture the complexity and 3D context of the tumor microenvironment and have consequently been implicated in the high percentage of drugs investigated in vitro that later fail in clinical trials. Herein, we screened two sophorolipid candidates and a clinically-used chemotherapeutic, doxorubicin, on in vitro breast cancer models ranging from 2D monolayers to 3D spheroids, employing optical coherence tomography to confirm these morphologies. We calculated corresponding IC50 values for these drugs and found one of the sophorolipids to have comparable toxicities to the chemotherapeutic control. Our findings show increased drug resistance associated with model dimensionality, such that all drugs tested showed that 3D spheroids exhibited higher IC50 values than their 2D counterparts. These findings demonstrate promising preliminary data to support the use of sophorolipids as a more affordable alternative to traditional clinical interventions and demonstrate the importance of 3D tumor models in assessing drug response.
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Affiliation(s)
- Cassandra L Roberge
- Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States
| | - Rebecca T Miceli
- Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180, United States
- Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, New York 12180, United States
| | - Lillian R Murphy
- Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States
| | - David M Kingsley
- Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States
| | - Richard A Gross
- Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180, United States
- Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, New York 12180, United States
| | - David T Corr
- Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States
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Karanfil AS, Louis F, Matsusaki M. Biofabrication of vascularized adipose tissues and their biomedical applications. MATERIALS HORIZONS 2023; 10:1539-1558. [PMID: 36789675 DOI: 10.1039/d2mh01391f] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/10/2023]
Abstract
Recent advances in adipose tissue engineering and cell biology have led to the development of innovative therapeutic strategies in regenerative medicine for adipose tissue reconstruction. To date, the many in vitro and in vivo models developed for vascularized adipose tissue engineering cover a wide range of research areas, including studies with cells of various origins and types, polymeric scaffolds of natural and synthetic derivation, models presented using decellularized tissues, and scaffold-free approaches. In this review, studies on adipose tissue types with different functions, characteristics and body locations have been summarized with 3D in vitro fabrication approaches. The reason for the particular focus on vascularized adipose tissue models is that current liposuction and fat transplantation methods are unsuitable for adipose tissue reconstruction as the lack of blood vessels results in inadequate nutrient and oxygen delivery, leading to necrosis in situ. In the first part of this paper, current studies and applications of white and brown adipose tissues are presented according to the polymeric materials used, focusing on the studies which could show vasculature in vitro and after in vivo implantation, and then the research on adipose tissue fabrication and applications are explained.
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Affiliation(s)
- Aslı Sena Karanfil
- Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Japan.
| | - Fiona Louis
- Joint Research Laboratory (TOPPAN) for Advanced Cell Regulatory Chemistry, Graduate School of Engineering, Osaka University, Japan
| | - Michiya Matsusaki
- Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Japan.
- Joint Research Laboratory (TOPPAN) for Advanced Cell Regulatory Chemistry, Graduate School of Engineering, Osaka University, Japan
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10
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Habra K, Pearson JRD, McArdle SEB. Robust formation of optimal single spheroids towards cost-effective in vitro three-dimensional tumor models. FEBS Open Bio 2023. [PMID: 37317692 DOI: 10.1002/2211-5463.13614] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2022] [Revised: 03/21/2023] [Accepted: 04/18/2023] [Indexed: 06/16/2023] Open
Abstract
While useful for fundamental in vitro studies, monolayer cell cultures are not physiologically relevant. Spheroids, a complex three-dimensional (3D) structure, more closely resemble in vivo tumor growth. Spheroids allow the results obtained relating to proliferation, cell death, differentiation, metabolism, and various antitumor therapies to be more predictive of in vivo outcomes. In the protocol herein, a rapid and high-throughput method is discussed for the generation of single spheroids using various cancer cell lines, including brain cancer cells (U87 MG, SEBTA-027, SF188), prostate cancer cells (DU-145, TRAMP-C1), and breast cancer cells (BT-549, Py230) in 96-round bottom-well plates. The proposed method is associated with significantly low costs per plate without requiring refining or transferring. Homogeneous compact spheroid morphology was evidenced as early as 1 day after following this protocol. Proliferating cells were traced in the rim, while dead cells were found to be located inside the core region of the spheroid using confocal microscopy and the Incucyte® live imaging system. H&E staining of spheroid sections was utilized to investigate the tightness of the cell packaging. Through western blotting analyses, it was revealed that a stem cell-like phenotype was adopted by these spheroids. This method was also used to obtain the EC50 of the anticancer dipeptide carnosine on U87 MG 3D culture. This affordable, easy-to-follow five-step protocol allows for the robust generation of various uniform spheroids with 3D morphology characteristics.
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Affiliation(s)
- Kinana Habra
- Chemistry department, School of Science and Technology, Nottingham Trent University, UK
- John van Geest Cancer Research Centre, School of Science and Technology, Nottingham Trent University, UK
| | - Joshua R D Pearson
- John van Geest Cancer Research Centre, School of Science and Technology, Nottingham Trent University, UK
- Centre for Health, Ageing and Understanding Disease, School of Science and Technology, Nottingham Trent University, UK
| | - Stéphanie E B McArdle
- John van Geest Cancer Research Centre, School of Science and Technology, Nottingham Trent University, UK
- Centre for Health, Ageing and Understanding Disease, School of Science and Technology, Nottingham Trent University, UK
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11
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Kieda J, Appak-Baskoy S, Jeyhani M, Navi M, Chan KWY, Tsai SSH. Microfluidically-generated Encapsulated Spheroids (μ-GELS): An All-Aqueous Droplet Microfluidics Platform for Multicellular Spheroids Generation. ACS Biomater Sci Eng 2023; 9:1043-1052. [PMID: 36626575 DOI: 10.1021/acsbiomaterials.2c00963] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Spheroids are three-dimensional clusters of cells that serve as in vitro tumor models to recapitulate in vivo morphology. A limitation of many existing on-chip platforms for spheroid formation is the use of cytotoxic organic solvents as the continuous phase in droplet generation processes. All-aqueous methods do not contain cytotoxic organic solvents but have so far been unable to achieve complete hydrogel gelation on chip. Here, we describe an enhanced droplet microfluidic platform that achieves on-chip gelation of all-aqueous hydrogel multicellular spheroids (MCSs). Specifically, we generate dextran-alginate droplets containing MCF-7 breast cancer cells, surrounded by polyethylene glycol, at a flow-focusing junction. Droplets then travel to a second flow-focusing junction where they interact with calcium chloride and gel on chip to form hydrogel MCSs. On-chip gelation of the MCSs is possible here because of an embedded capillary at the second junction that delays the droplet gelation, which prevents channel clogging problems that would otherwise exist. In drug-free experiments, we demonstrate that MCSs remain viable for 6 days. We also confirm the applicability of this system for cancer drug testing by observing that dose-dependent cell death is achievable using doxorubicin.
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Affiliation(s)
- Jennifer Kieda
- Graduate Program in Biomedical Engineering, Toronto Metropolitan University, TorontoM5B 2K3, Canada.,Keenan Research Centre for Biomedical Science, St. Michael's Hospital, TorontoM5B 2K3, Canada.,Institute for Biomedical Engineering, Science, and Technology (iBEST) - A partnership between Toronto Metropolitan University and St. Michael's Hospital, TorontoM5B 1W8, Canada
| | - Sila Appak-Baskoy
- Keenan Research Centre for Biomedical Science, St. Michael's Hospital, TorontoM5B 2K3, Canada.,Institute for Biomedical Engineering, Science, and Technology (iBEST) - A partnership between Toronto Metropolitan University and St. Michael's Hospital, TorontoM5B 1W8, Canada.,Department of Chemistry and Biology, Toronto Metropolitan University, TorontoM5B 2K3, Canada
| | - Morteza Jeyhani
- Keenan Research Centre for Biomedical Science, St. Michael's Hospital, TorontoM5B 2K3, Canada.,Institute for Biomedical Engineering, Science, and Technology (iBEST) - A partnership between Toronto Metropolitan University and St. Michael's Hospital, TorontoM5B 1W8, Canada.,Department of Mechanical and Industrial Engineering, Toronto Metropolitan University, TorontoM5B 2K3, Canada
| | - Maryam Navi
- Graduate Program in Biomedical Engineering, Toronto Metropolitan University, TorontoM5B 2K3, Canada.,Keenan Research Centre for Biomedical Science, St. Michael's Hospital, TorontoM5B 2K3, Canada.,Institute for Biomedical Engineering, Science, and Technology (iBEST) - A partnership between Toronto Metropolitan University and St. Michael's Hospital, TorontoM5B 1W8, Canada
| | - Katherine W Y Chan
- Graduate Program in Biomedical Engineering, Toronto Metropolitan University, TorontoM5B 2K3, Canada.,Keenan Research Centre for Biomedical Science, St. Michael's Hospital, TorontoM5B 2K3, Canada.,Institute for Biomedical Engineering, Science, and Technology (iBEST) - A partnership between Toronto Metropolitan University and St. Michael's Hospital, TorontoM5B 1W8, Canada
| | - Scott S H Tsai
- Graduate Program in Biomedical Engineering, Toronto Metropolitan University, TorontoM5B 2K3, Canada.,Keenan Research Centre for Biomedical Science, St. Michael's Hospital, TorontoM5B 2K3, Canada.,Institute for Biomedical Engineering, Science, and Technology (iBEST) - A partnership between Toronto Metropolitan University and St. Michael's Hospital, TorontoM5B 1W8, Canada.,Department of Mechanical and Industrial Engineering, Toronto Metropolitan University, TorontoM5B 2K3, Canada
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12
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Chipangura JK, Ntamo Y, Mohr B, Chellan N. A review of challenges and prospects of 3D cell-based culture models used for studying drug induced liver injury during early phases of drug development. Hum Exp Toxicol 2023; 42:9603271221147884. [PMID: 36879529 DOI: 10.1177/09603271221147884] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/08/2023]
Abstract
Drug-induced liver injury (DILI) is the leading cause of compound attrition during drug development. Over the years, a battery of in-vitro cell culture toxicity tests is being conducted to evaluate the toxicity of compounds prior to testing in laboratory animals. Two-dimensional (2D) in-vitro cell culture models are commonly used and have provided a great deal of knowledge; however, these models often fall short in mimicking natural structures of tissues in-vivo. Testing in humans is the most logical method, but unfortunately there are ethical limitations associated with human tests. To overcome these limitations better human-relevant, predictive models are required. The past decade has witnessed significant efforts towards the development of three-dimensional (3D) in-vitro cell culture models better mimicking in-vivo physiology. 3D cell culture has advantages in being representative of the interactions of cells in-vivo and when validated can act as an interphase between 2D cell culture models and in-vivo animal models. The current review seeks to provide an overview of the challenges that make biomarkers used for detection of DILI not to be sensitive enough during drug development and explore how 3D cell culture models can be used to address the gap with the current models.
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Affiliation(s)
- John K Chipangura
- Faculty of Health Science, University of Cape Town Research Animal Facility, South Africa
| | - Yonela Ntamo
- Biomedical Research and Innovation Platform, South African Medical Research Council, Tygerberg, South Africa
| | - Bert Mohr
- Faculty of Health Science, University of Cape Town Research Animal Facility, South Africa
| | - Nireshni Chellan
- Biomedical Research and Innovation Platform, South African Medical Research Council, Tygerberg, South Africa
- Centre for Cardio-metabolic Research in Africa (CARMA), Division of Medical Physiology, Faculty of Health Sciences, Stellenbosch University, South Africa
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13
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Tornes AJK, Stenberg VY, Larsen RH, Bruland ØS, Revheim ME, Juzeniene A. Targeted alpha therapy with the 224Ra/ 212Pb-TCMC-TP-3 dual alpha solution in a multicellular tumor spheroid model of osteosarcoma. Front Med (Lausanne) 2022; 9:1058863. [PMID: 36507500 PMCID: PMC9727293 DOI: 10.3389/fmed.2022.1058863] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2022] [Accepted: 11/04/2022] [Indexed: 11/24/2022] Open
Abstract
Osteosarcoma patients with overt metastases at primary diagnosis have a 5-year survival rate of less than 20%. TP-3 is a murine IgG2b monoclonal antibody with high affinity for an epitope residing on the p80 osteosarcoma cell surface membrane antigen. The tumor-associated antigen p80 is overexpressed in osteosarcomas, and has very low normal tissue expression. We propose a novel dual alpha targeting solution containing two radionuclides from the same decay chain, including the bone-seeking 224Ra, and cancer cell-surface seeking 212Pb-TCMC-TP-3 for the treatment of osteoblastic bone cancers, circulating cancer cells and micrometastases. In this in vitro study, the cytotoxic effects of 212Pb-TCMC-TP-3 (single alpha solution) and 224Ra/212Pb-TCMC-TP-3 (dual alpha solution) were investigated in a multicellular spheroid model mimicking micrometastatic disease in osteosarcoma. OHS spheroids with diameters of 253 ± 98 μm treated with 4.5, 2.7, and 3.3 kBq/ml of 212Pb-TCMC-TP-3 for 1, 4, and 24 h, respectively, were disintegrated within 3 weeks. The 212Pb-TCMC-TP-3 induced a 7-fold delay in spheroid doubling time compared to a 28-times higher dose with the non-specific 212Pb-TCMC-rituximab. The 224Ra/212Pb-TCMC-TP-3 completely disintegrated spheroids with diameters of 218-476 μm within 3 and 2 weeks after 4 and 24 h incubation with 5 kBq/ml, respectively. Treatment with 1 kBq/ml of 224Ra/212Pb-TCMC-TP-3 for 24 h caused an 11.4-fold reduction in spheroid viability compared with unconjugated 224Ra/212Pb. The single and dual alpha solutions with TP-3 showed cytotoxicity in spheroids of clinically relevant size, which warrant further testing of the dual alpha solution using in vivo osteosarcoma models.
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Affiliation(s)
- Anna Julie Kjøl Tornes
- Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway,ArtBio AS, Oslo, Norway,Institute of Clinical Medicine, University of Oslo, Oslo, Norway,*Correspondence: Anna Julie Kjøl Tornes,
| | - Vilde Yuli Stenberg
- Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway,ArtBio AS, Oslo, Norway,Institute of Clinical Medicine, University of Oslo, Oslo, Norway
| | | | - Øyvind Sverre Bruland
- Institute of Clinical Medicine, University of Oslo, Oslo, Norway,Department of Oncology, Oslo University Hospital, Oslo, Norway
| | - Mona-Elisabeth Revheim
- Institute of Clinical Medicine, University of Oslo, Oslo, Norway,Division of Radiology and Nuclear Medicine, Oslo University Hospital, Oslo, Norway
| | - Asta Juzeniene
- Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway,Department of Physics, University of Oslo, Oslo, Norway
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14
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Banerjee D, Singh YP, Datta P, Ozbolat V, O'Donnell A, Yeo M, Ozbolat IT. Strategies for 3D bioprinting of spheroids: A comprehensive review. Biomaterials 2022; 291:121881. [DOI: 10.1016/j.biomaterials.2022.121881] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2022] [Revised: 10/04/2022] [Accepted: 10/23/2022] [Indexed: 11/17/2022]
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15
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Jubelin C, Muñoz-Garcia J, Griscom L, Cochonneau D, Ollivier E, Heymann MF, Vallette FM, Oliver L, Heymann D. Three-dimensional in vitro culture models in oncology research. Cell Biosci 2022; 12:155. [PMID: 36089610 PMCID: PMC9465969 DOI: 10.1186/s13578-022-00887-3] [Citation(s) in RCA: 123] [Impact Index Per Article: 41.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2022] [Accepted: 08/18/2022] [Indexed: 11/10/2022] Open
Abstract
AbstractCancer is a multifactorial disease that is responsible for 10 million deaths per year. The intra- and inter-heterogeneity of malignant tumors make it difficult to develop single targeted approaches. Similarly, their diversity requires various models to investigate the mechanisms involved in cancer initiation, progression, drug resistance and recurrence. Of the in vitro cell-based models, monolayer adherent (also known as 2D culture) cell cultures have been used for the longest time. However, it appears that they are often less appropriate than the three-dimensional (3D) cell culture approach for mimicking the biological behavior of tumor cells, in particular the mechanisms leading to therapeutic escape and drug resistance. Multicellular tumor spheroids are widely used to study cancers in 3D, and can be generated by a multiplicity of techniques, such as liquid-based and scaffold-based 3D cultures, microfluidics and bioprinting. Organoids are more complex 3D models than multicellular tumor spheroids because they are generated from stem cells isolated from patients and are considered as powerful tools to reproduce the disease development in vitro. The present review provides an overview of the various 3D culture models that have been set up to study cancer development and drug response. The advantages of 3D models compared to 2D cell cultures, the limitations, and the fields of application of these models and their techniques of production are also discussed.
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16
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Rodriguez ME, Tekiel V, Campo VA. In vitro evaluation of Resveratrol as a potential pre-exposure prophylactic drug against Trypanosoma cruzi infection. Int J Parasitol Drugs Drug Resist 2022; 20:54-64. [PMID: 36099853 PMCID: PMC9474288 DOI: 10.1016/j.ijpddr.2022.08.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2022] [Revised: 07/19/2022] [Accepted: 08/15/2022] [Indexed: 12/14/2022]
Abstract
Chagas' disease or American trypanosomiasis, caused by Trypanosoma cruzi infection, is an endemic disease in Latin America, which has spread worldwide in the past years. The drugs presently used for treatment have shown limited efficacy due to the appearance of resistant parasites and severe side effects. Some of the most recent studies on anti-parasitic drugs have been focused on protein acetylation, a reversible reaction modulated by Acetyl Transferases (KATs) and Deacetylases (KDACs). We have previously reported the anti-parasite activity of resveratrol (RSV), an activator of KDACs type III (or sirtuins), and showed that this drug can reduce the growth of T. cruzi epimastigotes and the infectivity of trypomastigotes. Since RSV is now widely used in humans due to its beneficial effects as an antioxidant, it has become an attractive candidate as a repurposing drug. In this context, the aim of the present study was to evaluate the ability of this drug to protect three different types of host cells from parasite infection. RSV treatment before parasite infection reduced the percentage of infected cells by 50-70% depending on the cell type. Although the mammalian cell lines tested showed different sensitivity to RSV, apoptosis was not significantly affected, showing that RSV was able to protect cells from infection without the activation of this process. Since autophagy has been described as a key process in parasite invasion, we also monitored this process on host cells pretreated with RSV. The results showed that, at the concentrations and incubation times tested, autophagy was not induced in any of the cell types evaluated. Our results show a partial protective effect of RSV in vitro, which justifies extending studies to an in vivo model to elucidate the mechanism by which this effect occurs.
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Affiliation(s)
| | | | - Vanina A. Campo
- Corresponding author. IIB: Instituto de Investigaciones Biotecnologicas, Av. 25 de Mayo y Francia, 1650 San Martin, Buenos Aires, Argentina.
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17
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Evaluation of the Anti-Histoplasma capsulatum Activity of Indole and Nitrofuran Derivatives and Their Pharmacological Safety in Three-Dimensional Cell Cultures. Pharmaceutics 2022; 14:pharmaceutics14051043. [PMID: 35631629 PMCID: PMC9147190 DOI: 10.3390/pharmaceutics14051043] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Revised: 05/04/2022] [Accepted: 05/09/2022] [Indexed: 11/25/2022] Open
Abstract
Histoplasma capsulatum is a fungus that causes histoplasmosis. The increased evolution of microbial resistance and the adverse effects of current antifungals help new drugs to emerge. In this work, fifty-four nitrofurans and indoles were tested against the H. capsulatum EH-315 strain. Compounds with a minimum inhibitory concentration (MIC90) equal to or lower than 7.81 µg/mL were selected to evaluate their MIC90 on ATCC G217-B strain and their minimum fungicide concentration (MFC) on both strains. The quantification of membrane ergosterol, cell wall integrity, the production of reactive oxygen species, and the induction of death by necrosis–apoptosis was performed to investigate the mechanism of action of compounds 7, 11, and 32. These compounds could reduce the extracted sterol and induce necrotic cell death, similarly to itraconazole. Moreover, 7 and 11 damaged the cell wall, causing flaws in the contour (11), or changing the size and shape of the fungal cell wall (7). Furthermore, 7 and 32 induced reactive oxygen species (ROS) formation higher than 11 and control. Finally, the cytotoxicity was measured in two models of cell culture, i.e., monolayers (cells are flat) and a three-dimensional (3D) model, where they present a spheroidal conformation. Cytotoxicity assays in the 3D model showed a lower toxicity in the compounds than those performed on cell monolayers. Overall, these results suggest that derivatives of nitrofurans and indoles are promising compounds for the treatment of histoplasmosis.
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18
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Calori IR, Alves SR, Bi H, Tedesco AC. Type-I Collagen/Collagenase Modulates the 3D Structure and Behavior of Glioblastoma Spheroid Models. ACS APPLIED BIO MATERIALS 2022; 5:723-733. [PMID: 35068151 DOI: 10.1021/acsabm.1c01138] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Multicellular tumor spheroids have emerged as well-structured, three-dimensional culture models that resemble and mimic the complexity of the dense and hypoxic cancer microenvironment. However, in brain tumor studies, a variety of glioblastoma multiforme (GBM) cell lines only self-assemble into loose cellular aggregates, lacking the properties of actual glioma tumors in humans. In this study, we used type-I collagen as an extracellular matrix component to promote the compaction of GBM aggregates forming tight spheroids to understand how collagen influences the properties of tumors, such as their growth, proliferation, and invasion, and collagenase to promote collagen degradation. The GBM cell lines U87MG, T98G, and A172, as well as the medulloblastoma cell line UW473, were used as standard cell lines that do not spontaneously self-assemble into spheroids, and GBM U251 was used as a self-assembling cell line. According to the findings, all cell lines formed tight spheroids at collagen concentrations higher than 15.0 μg mL-1. Collagen was distributed along the spheroid, similarly to that observed in invasive GBM tumors, and decreased cell migration with no effect on the cellular uptake of small active molecules, as demonstrated by uptake studies using the photosensitizer verteporfin. The enzymatic cleavage of collagen affected spheroid morphology and increased cell migration while maintaining cell viability. Such behaviors are relevant to the physiological models of GBM tumors and are useful for better understanding cell migration and the in vivo infiltration path, drug screening, and kinetics of progression of GBM tumors.
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Affiliation(s)
- Italo Rodrigo Calori
- Department of Chemistry, Center of Nanotechnology and Tissue Engineering -Photobiology and Photomedicine Research Group, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo 14040-901, Brazil
| | - Samara Rodrigues Alves
- Department of Chemistry, Center of Nanotechnology and Tissue Engineering -Photobiology and Photomedicine Research Group, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo 14040-901, Brazil
| | - Hong Bi
- School of Chemistry and Chemical Engineering, Anhui Key Laboratory of Modern Biomanufacturing, Anhui University, Hefei 230601, China
| | - Antonio Claudio Tedesco
- Department of Chemistry, Center of Nanotechnology and Tissue Engineering -Photobiology and Photomedicine Research Group, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo 14040-901, Brazil.,School of Chemistry and Chemical Engineering, Anhui Key Laboratory of Modern Biomanufacturing, Anhui University, Hefei 230601, China
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19
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Ayvaz I, Sunay D, Sariyar E, Erdal E, Karagonlar ZF. Three-Dimensional Cell Culture Models of Hepatocellular Carcinoma - a Review. J Gastrointest Cancer 2021; 52:1294-1308. [PMID: 34927218 DOI: 10.1007/s12029-021-00772-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/16/2021] [Indexed: 01/09/2023]
Abstract
INTRODUCTION Three-dimensional (3D) cell culture studies are becoming extremely common because of their capability to mimic tumor architecture, such as cell-cell and cell-ECM interactions, more efficiently than 2D monolayer systems. These interactions have important roles in defining the tumor cell behaviors, such as proliferation, differentiation, and most importantly, tumor drug response. OBJECTIVE This review aims to provide an overview of the methods for 3D tumor spheroid formation to model human tumors, specifically concentrated on studies using hepatocellular carcinoma (HCC) cells. METHOD We obtained information from previously published articles. In this review, there is discussion of the scaffold and non-scaffold-based approaches, including hanging drop, bioreactors and 3D bioprinting. RESULTS AND CONCLUSION The mimicking of the tumor microenvironment (TME) as tumor spheroids could provide a valuable platform for studying tumor biology. Multicellular tumor spheroids are self-assembled cultures of mixed cells (tumor and stromal cells) organized in a 3D arrangement. These spheroids closely mimic the main features of human solid tumors, such as structural organization, central hypoxia, and overall oxygen and nutrient gradients. Hepatocellular carcinoma (HCC) is the most common liver malignancy, and most difficult to overcome because of its drug resistance and tumor heterogeneity. In order to mimic this highly heterogeneous environment, 3D cell culture systems are needed.
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Affiliation(s)
- Irmak Ayvaz
- Genetics and Bioengineering Department, Izmir University of Economics, Izmir, 35330, Turkey
| | - Dilara Sunay
- Genetics and Bioengineering Department, Izmir University of Economics, Izmir, 35330, Turkey
| | - Ece Sariyar
- Genetics and Bioengineering Department, Izmir University of Economics, Izmir, 35330, Turkey
| | - Esra Erdal
- Department of Medical Biology and Genetics, FacultyofMedicine, Dokuz Eylul University, Izmir, 35340, Turkey.,Izmir Biomedicine and Genome Center, Izmir, 35340, Turkey
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Ferreira L, Morais J, Preto M, Silva R, Urbatzka R, Vasconcelos V, Reis M. Uncovering the Bioactive Potential of a Cyanobacterial Natural Products Library Aided by Untargeted Metabolomics. Mar Drugs 2021; 19:633. [PMID: 34822504 PMCID: PMC8624515 DOI: 10.3390/md19110633] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2021] [Revised: 11/08/2021] [Accepted: 11/10/2021] [Indexed: 01/18/2023] Open
Abstract
The Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC) holds a vast number of cyanobacteria whose chemical richness is still largely unknown. To expedite its bioactivity screening we developed a natural products library. Sixty strains and four environmental samples were chromatographed, using a semiautomatic HPLC system, yielding 512 fractions that were tested for their cytotoxic activity against 2D and 3D models of human colon carcinoma (HCT 116), and non-cancerous cell line hCMEC/D3. Six fractions showed high cytotoxicity against 2D and 3D cell models (group A), and six other fractions were selected by their effects on 3D cells (group B). The metabolome of each group was organized and characterized using the MolNetEnhancer workflow, and its processing with MetaboAnalyst allowed discrimination of the mass features with the highest fold change, and thus the ones that might be bioactive. Of those, mass features without precedented identification were mostly found in group A, indicating seven possible novel bioactive molecules, alongside in silico putative annotation of five cytotoxic compounds. Manual dereplication of group B tentatively identified nine pheophytin and pheophorbide derivatives. Our approach enabled the selection of 7 out of 60 cyanobacterial strains for anticancer drug discovery, providing new data concerning the chemical composition of these cyanobacteria.
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Affiliation(s)
- Leonor Ferreira
- CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leixões, University of Porto, 4450-208 Matosinhos, Portugal; (L.F.); (J.M.); (M.P.); (R.S.); (R.U.); (V.V.)
| | - João Morais
- CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leixões, University of Porto, 4450-208 Matosinhos, Portugal; (L.F.); (J.M.); (M.P.); (R.S.); (R.U.); (V.V.)
- Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, Edifício FC4, 4169-007 Porto, Portugal
| | - Marco Preto
- CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leixões, University of Porto, 4450-208 Matosinhos, Portugal; (L.F.); (J.M.); (M.P.); (R.S.); (R.U.); (V.V.)
| | - Raquel Silva
- CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leixões, University of Porto, 4450-208 Matosinhos, Portugal; (L.F.); (J.M.); (M.P.); (R.S.); (R.U.); (V.V.)
| | - Ralph Urbatzka
- CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leixões, University of Porto, 4450-208 Matosinhos, Portugal; (L.F.); (J.M.); (M.P.); (R.S.); (R.U.); (V.V.)
| | - Vitor Vasconcelos
- CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leixões, University of Porto, 4450-208 Matosinhos, Portugal; (L.F.); (J.M.); (M.P.); (R.S.); (R.U.); (V.V.)
- Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, Edifício FC4, 4169-007 Porto, Portugal
| | - Mariana Reis
- CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leixões, University of Porto, 4450-208 Matosinhos, Portugal; (L.F.); (J.M.); (M.P.); (R.S.); (R.U.); (V.V.)
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21
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Habanjar O, Diab-Assaf M, Caldefie-Chezet F, Delort L. 3D Cell Culture Systems: Tumor Application, Advantages, and Disadvantages. Int J Mol Sci 2021; 22:12200. [PMID: 34830082 PMCID: PMC8618305 DOI: 10.3390/ijms222212200] [Citation(s) in RCA: 238] [Impact Index Per Article: 59.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2021] [Revised: 11/05/2021] [Accepted: 11/07/2021] [Indexed: 01/09/2023] Open
Abstract
The traditional two-dimensional (2D) in vitro cell culture system (on a flat support) has long been used in cancer research. However, this system cannot be fully translated into clinical trials to ideally represent physiological conditions. This culture cannot mimic the natural tumor microenvironment due to the lack of cellular communication (cell-cell) and interaction (cell-cell and cell-matrix). To overcome these limitations, three-dimensional (3D) culture systems are increasingly developed in research and have become essential for tumor research, tissue engineering, and basic biology research. 3D culture has received much attention in the field of biomedicine due to its ability to mimic tissue structure and function. The 3D matrix presents a highly dynamic framework where its components are deposited, degraded, or modified to delineate functions and provide a platform where cells attach to perform their specific functions, including adhesion, proliferation, communication, and apoptosis. So far, various types of models belong to this culture: either the culture based on natural or synthetic adherent matrices used to design 3D scaffolds as biomaterials to form a 3D matrix or based on non-adherent and/or matrix-free matrices to form the spheroids. In this review, we first summarize a comparison between 2D and 3D cultures. Then, we focus on the different components of the natural extracellular matrix that can be used as supports in 3D culture. Then we detail different types of natural supports such as matrigel, hydrogels, hard supports, and different synthetic strategies of 3D matrices such as lyophilization, electrospiding, stereolithography, microfluid by citing the advantages and disadvantages of each of them. Finally, we summarize the different methods of generating normal and tumor spheroids, citing their respective advantages and disadvantages in order to obtain an ideal 3D model (matrix) that retains the following characteristics: better biocompatibility, good mechanical properties corresponding to the tumor tissue, degradability, controllable microstructure and chemical components like the tumor tissue, favorable nutrient exchange and easy separation of the cells from the matrix.
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Affiliation(s)
- Ola Habanjar
- Université Clermont-Auvergne, INRAE, UNH, Unité de Nutrition Humaine, CRNH-Auvergne, 63000 Clermont-Ferrand, France; (O.H.); (F.C.-C.)
| | - Mona Diab-Assaf
- Equipe Tumorigénèse Pharmacologie Moléculaire et Anticancéreuse, Faculté des Sciences II, Université Libanaise Fanar, Beyrouth 1500, Liban;
| | - Florence Caldefie-Chezet
- Université Clermont-Auvergne, INRAE, UNH, Unité de Nutrition Humaine, CRNH-Auvergne, 63000 Clermont-Ferrand, France; (O.H.); (F.C.-C.)
| | - Laetitia Delort
- Université Clermont-Auvergne, INRAE, UNH, Unité de Nutrition Humaine, CRNH-Auvergne, 63000 Clermont-Ferrand, France; (O.H.); (F.C.-C.)
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22
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Gündel B, Liu X, Löhr M, Heuchel R. Pancreatic Ductal Adenocarcinoma: Preclinical in vitro and ex vivo Models. Front Cell Dev Biol 2021; 9:741162. [PMID: 34746135 PMCID: PMC8569794 DOI: 10.3389/fcell.2021.741162] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2021] [Accepted: 10/04/2021] [Indexed: 12/13/2022] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the most overlooked cancers despite its dismal median survival time of 6 months. The biggest challenges in improving patient survival are late diagnosis due to lack of diagnostic markers, and limited treatment options due to almost complete therapy resistance. The past decades of research identified the dense stroma and the complex interplay/crosstalk between the cancer- and the different stromal cells as the main culprits for the slow progress in improving patient outcome. For better ex vivo simulation of this complex tumor microenvironment the models used in PDAC research likewise need to become more diverse. Depending on the focus of the investigation, several in vitro and in vivo models for PDAC have been established in the past years. Particularly, 3D cell culture such as spheroids and organoids have become more frequently used. This review aims to examine current PDAC in vitro models, their inherent limitations, and their successful implementations in research.
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Affiliation(s)
- Beate Gündel
- Pancreas Cancer Research Lab, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Huddinge, Sweden
| | - Xinyuan Liu
- Pancreas Cancer Research Lab, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Huddinge, Sweden
| | - Matthias Löhr
- Pancreas Cancer Research Lab, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Huddinge, Sweden
- Department of Upper GI, C1:77, Karolinska Comprehensive Cancer Center, Stockholm, Sweden
| | - Rainer Heuchel
- Pancreas Cancer Research Lab, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Huddinge, Sweden
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23
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Jauković A, Abadjieva D, Trivanović D, Stoyanova E, Kostadinova M, Pashova S, Kestendjieva S, Kukolj T, Jeseta M, Kistanova E, Mourdjeva M. Specificity of 3D MSC Spheroids Microenvironment: Impact on MSC Behavior and Properties. Stem Cell Rev Rep 2021; 16:853-875. [PMID: 32681232 DOI: 10.1007/s12015-020-10006-9] [Citation(s) in RCA: 67] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Mesenchymal stem cells (MSC) have been considered the promising candidates for the regenerative and personalized medicine due to their self-renewal potential, multilineage differentiation and immunomodulatory capacity. Although these properties have encouraged profound MSC studies in recent years, the majority of research has been based on standard 2D culture utilization. The opportunity to resemble in vivo characteristics of cells native niche has been provided by implementation of 3D culturing models such as MSC spheroid formation assesed through cells self-assembling. In this review, we address the current literature on physical and biochemical features of 3D MSC spheroid microenvironment and their impact on MSC properties and behaviors. Starting with the reduction in the cells' dimensions and volume due to the changes in adhesion molecules expression and cytoskeletal proteins rearrangement resembling native conditions, through the microenvironment shifts in oxygen, nutrients and metabolites gradients and demands, we focus on distinctive and beneficial features of MSC in spheroids compared to cells cultured in 2D conditions. By summarizing the data for 3D MSC spheroids regarding cell survival, pluripotency, differentiation, immunomodulatory activities and potential to affect tumor cells growth we highlighted advantages and perspectives of MSC spheroids use in regenerative medicine. Further detailed analyses are needed to deepen our understanding of mechanisms responsible for modified MSC behavior in spheroids and to set future directions for MSC clinical application.
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Affiliation(s)
- Aleksandra Jauković
- Laboratory for Experimental Hematology and Stem Cells, Institute for Medical Research, University of Belgrade, Dr. Subotića 4, PO BOX 102, Belgrade, 11129, Serbia
| | - Desislava Abadjieva
- Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, 73 Tzarigradsko shoes, 1113, Sofia, Bulgaria
| | - Drenka Trivanović
- Laboratory for Experimental Hematology and Stem Cells, Institute for Medical Research, University of Belgrade, Dr. Subotića 4, PO BOX 102, Belgrade, 11129, Serbia.,IZKF Group Tissue Regeneration in Musculoskeletal Diseases, University Clinics, Röntgenring 11, D-97070, Wuerzburg, Germany.,Bernhard-Heine-Center for Locomotion Research, University Wuerzburg, Wuerzburg, Germany
| | - Elena Stoyanova
- Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, 73 Tzarigradsko shoes, 1113, Sofia, Bulgaria
| | - Milena Kostadinova
- Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, 73 Tzarigradsko shoes, 1113, Sofia, Bulgaria
| | - Shina Pashova
- Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, 73 Tzarigradsko shoes, 1113, Sofia, Bulgaria
| | - Snejana Kestendjieva
- Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, 73 Tzarigradsko shoes, 1113, Sofia, Bulgaria
| | - Tamara Kukolj
- Laboratory for Experimental Hematology and Stem Cells, Institute for Medical Research, University of Belgrade, Dr. Subotića 4, PO BOX 102, Belgrade, 11129, Serbia
| | - Michal Jeseta
- Department of Obstetrics and Gynecology, University Hospital and Masaryk University, Obilní trh 11, 602 00, Brno, Czech Republic.,Department of Veterinary Sciences, Czech University of Life Sciences in Prague, Kamýcká 129, 165 00, Suchdol, Praha 6, Czech Republic
| | - Elena Kistanova
- Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, 73 Tzarigradsko shoes, 1113, Sofia, Bulgaria
| | - Milena Mourdjeva
- Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, 73 Tzarigradsko shoes, 1113, Sofia, Bulgaria.
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24
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Coltman NJ, Coke BA, Chatzi K, Shepherd EL, Lalor PF, Schulz-Utermoehl T, Hodges NJ. Application of HepG2/C3A liver spheroids as a model system for genotoxicity studies. Toxicol Lett 2021; 345:34-45. [PMID: 33865918 DOI: 10.1016/j.toxlet.2021.04.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Revised: 03/19/2021] [Accepted: 04/11/2021] [Indexed: 12/14/2022]
Abstract
HepG2 cells continue to be a valuable tool in early drug discovery and pharmaceutical development. In the current study we develop a 3D in vitro liver model, using HepG2/C3A cells that is predictive of human genotoxic exposure. HepG2/C3A cells cultured for 7-days in agarose-coated microplates formed spheroids which were uniform in shape and had well defined outer perimeters and no evidence of a hypoxic core. Quantitative real-time-PCR analysis showed statistically significant transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, UG1A1, UGT1A3, UGT1A6, EPHX, NAT2) and genes linked to liver function (ALB, CAR) in 3D cultures. In response to three model pro-genotoxicants: benzo[a]pyrene, amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-aminoanthracene (2-AA), we observed further transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, NAT1/2, SULT1A2, UGT1A1, UGT1A3) compared to untreated spheroids. Consistent with this, spheroids were more sensitive than 2D monolayers to compound induced single- and double- stranded DNA-damage as assessed by the comet assay and γH2AX phosphorylation respectively. In contrast, levels of DNA-damage induced by the direct acting mutagen 4-nitroquinoline N-oxide (4NQO) was the same in spheroids and monolayers. In support of the enhanced genotoxic response in spheroids we also observed transcriptional upregulation of genes relating to DNA-damage and cellular stress response (e.g. GADD45A and CDKN1A) in spheroids. In conclusion, HepG2/C3A 3D spheroids are a sensitive model for in vitro genotoxicity assessment with potential applications in early stage drug development.
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Affiliation(s)
- Nicholas J Coltman
- School of Biosciences, University of Birmingham, Birmingham, B15 2TT, United Kingdom.
| | - Brandon A Coke
- School of Biosciences, University of Birmingham, Birmingham, B15 2TT, United Kingdom
| | - Kyriaki Chatzi
- School of Biosciences, University of Birmingham, Birmingham, B15 2TT, United Kingdom
| | - Emma L Shepherd
- Centre for Liver and Gastrointestinal Research, Institute of Immunology and Inflammation, and National Institute for Health Research (NIHR) Birmingham Biomedical Research Centre, The Medical School, University of Birmingham, Birmingham, B15 2TT, United Kingdom
| | - Patricia F Lalor
- Centre for Liver and Gastrointestinal Research, Institute of Immunology and Inflammation, and National Institute for Health Research (NIHR) Birmingham Biomedical Research Centre, The Medical School, University of Birmingham, Birmingham, B15 2TT, United Kingdom
| | - Timothy Schulz-Utermoehl
- Sygnature Discovery, The Discovery Building, BioCity, Pennyfoot Street, Nottingham, United Kingdom
| | - Nikolas J Hodges
- School of Biosciences, University of Birmingham, Birmingham, B15 2TT, United Kingdom.
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25
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Abreu TR, Biscaia M, Gonçalves N, Fonseca NA, Moreira JN. In Vitro and In Vivo Tumor Models for the Evaluation of Anticancer Nanoparticles. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1295:271-299. [PMID: 33543464 DOI: 10.1007/978-3-030-58174-9_12] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Multiple studies about tumor biology have revealed the determinant role of the tumor microenvironment in cancer progression, resulting from the dynamic interactions between tumor cells and surrounding stromal cells within the extracellular matrix. This malignant microenvironment highly impacts the efficacy of anticancer nanoparticles by displaying drug resistance mechanisms, as well as intrinsic physical and biochemical barriers, which hamper their intratumoral accumulation and biological activity.Currently, two-dimensional cell cultures are used as the initial screening method in vitro for testing cytotoxic nanocarriers. However, this fails to mimic the tumor heterogeneity, as well as the three-dimensional tumor architecture and pathophysiological barriers, leading to an inaccurate pharmacological evaluation.Biomimetic 3D in vitro tumor models, on the other hand, are emerging as promising tools for more accurately assessing nanoparticle activity, owing to their ability to recapitulate certain features of the tumor microenvironment and thus provide mechanistic insights into nanocarrier intratumoral penetration and diffusion rates.Notwithstanding, in vivo validation of nanomedicines remains irreplaceable at the preclinical stage, and a vast variety of more advanced in vivo tumor models is currently available. Such complex animal models (e.g., genetically engineered mice and patient-derived xenografts) are capable of better predicting nanocarrier clinical efficiency, as they closely resemble the heterogeneity of the human tumor microenvironment.Herein, the development of physiologically more relevant in vitro and in vivo tumor models for the preclinical evaluation of anticancer nanoparticles will be discussed, as well as the current limitations and future challenges in clinical translation.
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Affiliation(s)
- Teresa R Abreu
- CNC - Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Faculty of Medicine (Polo 1), Rua Larga, Coimbra, Portugal.,UC - University of Coimbra, CIBB, Faculty of Pharmacy, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, Coimbra, Portugal
| | - Mariana Biscaia
- CNC - Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Faculty of Medicine (Polo 1), Rua Larga, Coimbra, Portugal
| | - Nélio Gonçalves
- CNC - Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Faculty of Medicine (Polo 1), Rua Larga, Coimbra, Portugal
| | - Nuno A Fonseca
- CNC - Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Faculty of Medicine (Polo 1), Rua Larga, Coimbra, Portugal.,TREAT U, SA, Parque Industrial de Taveiro, Lote 44, Coimbra, Portugal
| | - João Nuno Moreira
- CNC - Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Faculty of Medicine (Polo 1), Rua Larga, Coimbra, Portugal. .,UC - University of Coimbra, CIBB, Faculty of Pharmacy, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, Coimbra, Portugal.
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26
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Saleh NA, Rode MP, Sierra JA, Silva AH, Miyake JA, Filippin-Monteiro FB, Creczynski-Pasa TB. Three-dimensional multicellular cell culture for anti-melanoma drug screening: focus on tumor microenvironment. Cytotechnology 2020; 73:35-48. [PMID: 33505112 DOI: 10.1007/s10616-020-00440-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2020] [Accepted: 11/04/2020] [Indexed: 12/12/2022] Open
Abstract
Abstract The development of new treatments for malignant melanoma, which has the worst prognosis among skin neoplasms, remains a challenge. The tumor microenvironment aids tumor cells to grow and resist to chemotherapeutic treatment. One way to mimic and study the tumor microenvironment is by using three-dimensional (3D) co-culture models (spheroids). In this study, a melanoma heterospheroid model composed of cancer cells, fibroblasts, and macrophages was produced by liquid-overlay technique using the agarose gel. The size, growth, viability, morphology, cancer stem-like cells population and inflammatory profile of tumor heterospheroids and monospheroids were analyzed to evaluate the influence of stromal cells on these parameters. Furthermore, dacarbazine cytotoxicity was evaluated using spheroids and two-dimensional (2D) melanoma model. After finishing the experiments, it was observed the M2 macrophages induced an anti-inflammatory microenvironment in heterospheroids; fibroblasts cells support the formation of the extracellular matrix, and a higher percentage of melanoma CD271 was observed in this model. Additionally, melanoma spheroids responded differently to the dacarbazine than the 2D melanoma culture as a result of their cellular heterogeneity and 3D structure. The 3D model was shown to be a fast and reliable tool for drug screening, which can mimic the in vivo tumor microenvironment regarding interactions and complexity. Graphic abstract
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Affiliation(s)
- Najla Adel Saleh
- Departamento de Ciências Farmacêuticas, GEIMM-Grupo de Estudos de Interações entre Micro e Macromoléculas, Universidade Federal de Santa Catarina, S/N Centro de Ciências da Saúde Bloco H - 3° andar, sala H302-Bairro Trindade, Florianópolis, Santa Catarina CEP: 88040-900 Brazil
| | - Michele Patrícia Rode
- Departamento de Ciências Farmacêuticas, GEIMM-Grupo de Estudos de Interações entre Micro e Macromoléculas, Universidade Federal de Santa Catarina, S/N Centro de Ciências da Saúde Bloco H - 3° andar, sala H302-Bairro Trindade, Florianópolis, Santa Catarina CEP: 88040-900 Brazil
| | | | - Adny Henrique Silva
- Departamento de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC Brazil
| | - Juliano Andreoli Miyake
- Departamento de Ciências Morfológicas, Universidade Federal de Santa Catarina, Florianópolis, SC Brazil
| | - Fabíola Branco Filippin-Monteiro
- Departamento de Análises Clínicas, GEIMM-Grupo de Estudos de Interações entre Micro e Macromoléculas, Universidade Federal de Santa Catarina, S/N Centro de Ciências da Saúde Bloco H - 3° andar, sala H302-Bairro Trindade, Florianópolis, Santa Catarina CEP: 88040-900 Brazil
| | - Tânia Beatriz Creczynski-Pasa
- Departamento de Ciências Farmacêuticas, GEIMM-Grupo de Estudos de Interações entre Micro e Macromoléculas, Universidade Federal de Santa Catarina, S/N Centro de Ciências da Saúde Bloco H - 3° andar, sala H302-Bairro Trindade, Florianópolis, Santa Catarina CEP: 88040-900 Brazil
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27
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Roberge CL, Kingsley DM, Faulkner DE, Sloat CJ, Wang L, Barroso M, Intes X, Corr DT. Non-Destructive Tumor Aggregate Morphology and Viability Quantification at Cellular Resolution, During Development and in Response to Drug. Acta Biomater 2020; 117:322-334. [PMID: 33007490 DOI: 10.1016/j.actbio.2020.09.042] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2020] [Revised: 09/15/2020] [Accepted: 09/22/2020] [Indexed: 12/15/2022]
Abstract
Three-dimensional (3D) tissue-engineered in vitro models, particularly multicellular spheroids and organoids, have become important tools to explore disease progression and guide the development of novel therapeutic strategies. These avascular constructs are particularly powerful in oncological research due to their ability to mimic several key aspects of in vivo tumors, such as 3D structure and pathophysiologic gradients. Advancement of spheroid models requires characterization of critical features (i.e., size, shape, cellular density, and viability) during model development, and in response to treatment. However, evaluation of these characteristics longitudinally, quantitatively and non-invasively remains a challenge. Herein, Optical Coherence Tomography (OCT) is used as a label-free tool to assess 3D morphologies and cellular densities of tumor spheroids generated via the liquid overlay technique. We utilize this quantitative tool to assess Matrigel's influence on spheroid morphologic development, finding that the absence of Matrigel produces flattened, disk-like aggregates rather than 3D spheroids with physiologically-relevant features. Furthermore, this technology is adapted to quantify cell number within tumor spheroids, and to discern between live and dead cells, to non-destructively provide valuable information on tissue/construct viability, as well as a proof-of-concept for longitudinal drug efficacy studies. Together, these findings demonstrate OCT as a promising noninvasive, quantitative, label-free, longitudinal and cell-based method that can assess development and drug response in 3D cellular aggregates at a mesoscopic scale.
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Affiliation(s)
- Cassandra L Roberge
- Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 110 Eighth St., Troy, NY 12180, USA.
| | - David M Kingsley
- Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 110 Eighth St., Troy, NY 12180, USA.
| | - Denzel E Faulkner
- Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 110 Eighth St., Troy, NY 12180, USA.
| | - Charles J Sloat
- Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 110 Eighth St., Troy, NY 12180, USA.
| | - Ling Wang
- Department of Molecular and Cellular Physiology, Albany Medical College, Albany, NY, 12208, USA.
| | - Margarida Barroso
- Department of Molecular and Cellular Physiology, Albany Medical College, Albany, NY, 12208, USA.
| | - Xavier Intes
- Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 110 Eighth St., Troy, NY 12180, USA.
| | - David T Corr
- Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 110 Eighth St., Troy, NY 12180, USA.
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28
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Rodríguez ME, Rizzi M, Caeiro LD, Masip YE, Perrone A, Sánchez DO, Búa J, Tekiel V. Transmigration of Trypanosoma cruzi trypomastigotes through 3D cultures resembling a physiological environment. Cell Microbiol 2020; 22:e13207. [PMID: 32270902 DOI: 10.1111/cmi.13207] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2019] [Revised: 03/05/2020] [Accepted: 03/29/2020] [Indexed: 12/24/2022]
Abstract
To disseminate and colonise tissues in the mammalian host, Trypanosoma cruzi trypomastogotes should cross several biological barriers. How this process occurs or its impact in the outcome of the disease is largely speculative. We examined the in vitro transmigration of trypomastigotes through three-dimensional cultures (spheroids) to understand the tissular dissemination of different T. cruzi strains. Virulent strains were highly invasive: trypomastigotes deeply transmigrate up to 50 μm inside spheroids and were evenly distributed at the spheroid surface. Parasites inside spheroids were systematically observed in the space between cells suggesting a paracellular route of transmigration. On the contrary, poorly virulent strains presented a weak migratory capacity and remained in the external layers of spheroids with a patch-like distribution pattern. The invasiveness-understood as the ability to transmigrate deep into spheroids-was not a transferable feature between strains, neither by soluble or secreted factors nor by co-cultivation of trypomastigotes from invasive and non-invasive strains. Besides, we demonstrated that T. cruzi isolates from children that were born congenitally infected presented a highly migrant phenotype while an isolate from an infected mother (that never transmitted the infection to any of her children) presented significantly less migration. In brief, we demonstrated that in a 3D microenvironment each strain presents a characteristic migration pattern that can be associated to their in vivo behaviour. Altogether, data presented here repositionate spheroids as a valuable tool to study host-pathogen interactions.
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Affiliation(s)
- Matías Exequiel Rodríguez
- Instituto de Investigaciones Biotecnológicas "Dr. R. Ugalde" (IIBIO) Universidad Nacional de San Martín (UNSAM)-CONICET, Buenos Aires, Argentina
| | - Mariana Rizzi
- Instituto de Investigaciones Biotecnológicas "Dr. R. Ugalde" (IIBIO) Universidad Nacional de San Martín (UNSAM)-CONICET, Buenos Aires, Argentina
| | - Lucas D Caeiro
- Instituto de Investigaciones Biotecnológicas "Dr. R. Ugalde" (IIBIO) Universidad Nacional de San Martín (UNSAM)-CONICET, Buenos Aires, Argentina
| | - Yamil E Masip
- Instituto de Investigaciones Biotecnológicas "Dr. R. Ugalde" (IIBIO) Universidad Nacional de San Martín (UNSAM)-CONICET, Buenos Aires, Argentina
| | - Alina Perrone
- Instituto Nacional de Parasitología "Dr Mario Fatala Chaben", ANLIS-Carlos G. Malbrán, Buenos Aires, Argentina
| | - Daniel O Sánchez
- Instituto de Investigaciones Biotecnológicas "Dr. R. Ugalde" (IIBIO) Universidad Nacional de San Martín (UNSAM)-CONICET, Buenos Aires, Argentina
| | - Jacqueline Búa
- Instituto Nacional de Parasitología "Dr Mario Fatala Chaben", ANLIS-Carlos G. Malbrán, Buenos Aires, Argentina
| | - Valeria Tekiel
- Instituto de Investigaciones Biotecnológicas "Dr. R. Ugalde" (IIBIO) Universidad Nacional de San Martín (UNSAM)-CONICET, Buenos Aires, Argentina
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29
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Chaicharoenaudomrung N, Kunhorm P, Noisa P. Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling. World J Stem Cells 2019; 11:1065-1083. [PMID: 31875869 PMCID: PMC6904866 DOI: 10.4252/wjsc.v11.i12.1065] [Citation(s) in RCA: 239] [Impact Index Per Article: 39.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/26/2019] [Revised: 10/09/2019] [Accepted: 11/05/2019] [Indexed: 02/06/2023] Open
Abstract
Three-dimensional (3D) culture systems are becoming increasingly popular due to their ability to mimic tissue-like structures more effectively than the monolayer cultures. In cancer and stem cell research, the natural cell characteristics and architectures are closely mimicked by the 3D cell models. Thus, the 3D cell cultures are promising and suitable systems for various proposes, ranging from disease modeling to drug target identification as well as potential therapeutic substances that may transform our lives. This review provides a comprehensive compendium of recent advancements in culturing cells, in particular cancer and stem cells, using 3D culture techniques. The major approaches highlighted here include cell spheroids, hydrogel embedding, bioreactors, scaffolds, and bioprinting. In addition, the progress of employing 3D cell culture systems as a platform for cancer and stem cell research was addressed, and the prominent studies of 3D cell culture systems were discussed.
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Affiliation(s)
- Nipha Chaicharoenaudomrung
- Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
| | - Phongsakorn Kunhorm
- Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
| | - Parinya Noisa
- Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
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30
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Spheroid Culture System Methods and Applications for Mesenchymal Stem Cells. Cells 2019; 8:cells8121620. [PMID: 31842346 PMCID: PMC6953111 DOI: 10.3390/cells8121620] [Citation(s) in RCA: 300] [Impact Index Per Article: 50.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2019] [Revised: 12/09/2019] [Accepted: 12/09/2019] [Indexed: 12/16/2022] Open
Abstract
Owing to the importance of stem cell culture systems in clinical applications, researchers have extensively studied them to optimize the culture conditions and increase efficiency of cell culture. A spheroid culture system provides a similar physicochemical environment in vivo by facilitating cell–cell and cell–matrix interaction to overcome the limitations of traditional monolayer cell culture. In suspension culture, aggregates of adjacent cells form a spheroid shape having wide utility in tumor and cancer research, therapeutic transplantation, drug screening, and clinical study, as well as organic culture. There are various spheroid culture methods such as hanging drop, gel embedding, magnetic levitation, and spinner culture. Lately, efforts are being made to apply the spheroid culture system to the study of drug delivery platforms and co-cultures, and to regulate differentiation and pluripotency. To study spheroid cell culture, various kinds of biomaterials are used as building forms of hydrogel, film, particle, and bead, depending upon the requirement. However, spheroid cell culture system has limitations such as hypoxia and necrosis in the spheroid core. In addition, studies should focus on methods to dissociate cells from spheroid into single cells.
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Wörsdörfer P, Dalda N, Kern A, Krüger S, Wagner N, Kwok CK, Henke E, Ergün S. Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells. Sci Rep 2019; 9:15663. [PMID: 31666641 PMCID: PMC6821804 DOI: 10.1038/s41598-019-52204-7] [Citation(s) in RCA: 148] [Impact Index Per Article: 24.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2019] [Accepted: 10/14/2019] [Indexed: 12/19/2022] Open
Abstract
Organoids derived from human pluripotent stem cells are interesting models to study mechanisms of morphogenesis and promising platforms for disease modeling and drug screening. However, they mostly remain incomplete as they lack stroma, tissue resident immune cells and in particular vasculature, which create important niches during development and disease. We propose, that the directed incorporation of mesodermal progenitor cells (MPCs) into organoids will overcome the aforementioned limitations. In order to demonstrate the feasibility of the method, we generated complex human tumor as well as neural organoids. We show that the formed blood vessels display a hierarchic organization and mural cells are assembled into the vessel wall. Moreover, we demonstrate a typical blood vessel ultrastructure including endothelial cell-cell junctions, a basement membrane as well as luminal caveolae and microvesicles. We observe a high plasticity in the endothelial network, which expands, while the organoids grow and is responsive to anti-angiogenic compounds and pro-angiogenic conditions such as hypoxia. We show that vessels within tumor organoids connect to host vessels following transplantation. Remarkably, MPCs also deliver Iba1+ cells that infiltrate the neural tissue in a microglia-like manner.
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Affiliation(s)
- Philipp Wörsdörfer
- Institute of Anatomy and Cell Biology, Koellikerstraße 6, University of Würzburg, 97070, Würzburg, Germany.
| | - Nahide Dalda
- Institute of Anatomy and Cell Biology, Koellikerstraße 6, University of Würzburg, 97070, Würzburg, Germany
| | - Anna Kern
- Institute of Anatomy and Cell Biology, Koellikerstraße 6, University of Würzburg, 97070, Würzburg, Germany
| | - Sarah Krüger
- Institute of Anatomy and Cell Biology, Koellikerstraße 6, University of Würzburg, 97070, Würzburg, Germany
| | - Nicole Wagner
- Institute of Anatomy and Cell Biology, Koellikerstraße 6, University of Würzburg, 97070, Würzburg, Germany
| | - Chee Keong Kwok
- Institute of Anatomy and Cell Biology, Koellikerstraße 6, University of Würzburg, 97070, Würzburg, Germany
| | - Erik Henke
- Institute of Anatomy and Cell Biology, Koellikerstraße 6, University of Würzburg, 97070, Würzburg, Germany
| | - Süleyman Ergün
- Institute of Anatomy and Cell Biology, Koellikerstraße 6, University of Würzburg, 97070, Würzburg, Germany
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Palazzo E, Marconi A, Pincelli C, Morasso MI. Do DLX3 and CD271 Protect Human Keratinocytes from Squamous Tumor Development? Int J Mol Sci 2019; 20:ijms20143541. [PMID: 31331058 PMCID: PMC6678400 DOI: 10.3390/ijms20143541] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2019] [Revised: 07/15/2019] [Accepted: 07/18/2019] [Indexed: 11/29/2022] Open
Abstract
Well-regulated epidermal homeostasis depends on the function of different classes of factors, such as transcription regulators and receptors. Alterations in this homeostatic balance may lead to the development of cutaneous squamous tumorigenesis. The homeobox transcription factor DLX3 is determinant for a p53-dependent regulation of epidermal differentiation and modulates skin carcinogenesis. The maintenance of skin homeostasis also involves the action of neurotrophins (NTs) and their receptors, Trk and CD271. While Trk receptor overexpression is a hallmark of cancer, there are conflicting data on CD271 expression and function in cutaneous SCC (cSCC). Previous studies have reported NT receptors expression in head and neck SSC (HNSCC). We show that CD271 is expressed at low levels in primary cSCC cells and the number of CD271+ cells correlates with cell cohesion in SCC spheroids. In normal epidermis, CD271 is expressed in proliferative progenitor cells and DLX3 in terminally differentiated keratinocytes. Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) increase DLX3 expression. In the absence of a functional BDNF receptor TrkB in keratinocytes, we hypothesize that the BDNF-dependent DLX3 response could be mediated via CD271. Altogether, our results support a putative CD271-DLX3 connection in keratinocytes, which might be crucial to preventing squamous skin cancer.
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Affiliation(s)
- Elisabetta Palazzo
- Laboratory of Cutaneous Biology, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, 41100 Modena, Italy.
| | - Alessandra Marconi
- Laboratory of Cutaneous Biology, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, 41100 Modena, Italy
| | - Carlo Pincelli
- Laboratory of Cutaneous Biology, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, 41100 Modena, Italy
| | - Maria I Morasso
- Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Light sheet fluorescence microscopy versus confocal microscopy: in quest of a suitable tool to assess drug and nanomedicine penetration into multicellular tumor spheroids. Eur J Pharm Biopharm 2019; 142:195-203. [PMID: 31228557 DOI: 10.1016/j.ejpb.2019.06.019] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2019] [Revised: 05/23/2019] [Accepted: 06/17/2019] [Indexed: 02/01/2023]
Abstract
We recently constructed a multicellular spheroid model of pancreatic tumor based on a triple co-culture of cancer cells, fibroblasts and endothelial cells and characterized by the presence of fibronectin, an important component of the tumor extracellular matrix. By combining cancer cells and stromal components, this model recreates in vitro the three-dimensional (3D) architecture of solid tumors. In this study, we used these hetero-type spheroids as a tool to assess the penetration of doxorubicin (used as a model drug) through the whole tumor mass either in a free form or loaded into polymer nanoparticles (NPs), and we investigated whether microscopy images, acquired by Confocal Laser Scanning Microscopy (CLSM) and Light Sheet Fluorescence Microscopy (LSFM), would be best to provide reliable information on this process. Results clearly demonstrated that CLSM was not suitable to accurately monitor the diffusion of small molecules such as the doxorubicin. Indeed, it only allowed to scan a layer of 100 µm depth and no information on deeper layers could be available because of a progressive loss of the fluorescence signal. On the contrary, a complete 3D tomography of the hetero-type multicellular tumor spheroids (MCTS) was obtained by LSFM and multi-view image fusion which revealed that the fluorescent molecule was able to reach the core of spheroids as large as 1 mm in diameter. However, no doxorubicin-loaded polymer nanoparticles were detected in the spheroids, highlighting the challenge of nanomedicine delivery through biological barriers. Overall, the combination of hetero-type MCTS and LSFM allowed to carry out a highly informative microscopic assessment and represents a suitable approach to precisely follow up the drug penetration in tumors. Accordingly, it could provide useful support in the preclinical investigation and optimization of nanoscale systems for drug delivery to solid tumors.
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Nanomechanical Analysis of Extracellular Matrix and Cells in Multicellular Spheroids. Cell Mol Bioeng 2019; 12:203-214. [PMID: 31719910 DOI: 10.1007/s12195-019-00577-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2018] [Accepted: 05/20/2019] [Indexed: 01/09/2023] Open
Abstract
Introduction Over the last decade, atomic force microscopy (AFM) has played an important role in understanding nanomechanical properties of various cancer cell lines. This study is focused on Lewis lung carcinoma cell tumours as 3D multicellular spheroid (MS). Not much is know about the mechanical properties of the cells and the surrounding extracellular matrix (ECM) in rapidly growing tumours. Methods Depth-dependent indentation measurements were conducted with the AFM. Force-vs.-indentation curves were used to create stiffness profiles as a function of depth. Here studies were focused on the outer most layer, i.e., proliferation zone of the spheroid. Results Both surface and sub-surface stiffness profiles of MS were created. This study revealed three nanomechanical topographies, Type A-high modulus due to collagen fibers, Type B-high stiffness at cell membrane and ECM interface and Type C-increased modulus due to cell lying deep inside matrix at a depth of 1.35 μm. Both Type and Type-B topographies result from collagen-based structures in ECM. Conclusion This study has first time revealed mechanical constitution of an MS. Depth-dependent indentation studies have the revealed role of various molecular and cellular components responsible for providing mechanical stability to MS. Nanomechanical heterogeneities revealed in this investigation can shed new light in developing correct dosage regime for collagenase treatment of tumours and designing better controlled artificial extracellular matrix systems for replicating tissue growth in-vitro.
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Alhaque S, Themis M, Rashidi H. Three-dimensional cell culture: from evolution to revolution. Philos Trans R Soc Lond B Biol Sci 2019; 373:rstb.2017.0216. [PMID: 29786551 DOI: 10.1098/rstb.2017.0216] [Citation(s) in RCA: 49] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/19/2018] [Indexed: 02/06/2023] Open
Abstract
Recent advances in the isolation of tissue-resident adult stem cells and the identification of inductive factors that efficiently direct differentiation of human pluripotent stem cells along specific lineages have facilitated the development of high-fidelity modelling of several tissues in vitro Many of the novel approaches have employed self-organizing three-dimensional (3D) culturing of organoids, which offer several advantages over conventional two-dimensional platforms. Organoid technologies hold great promise for modelling diseases and predicting the outcome of drug responses in vitro Here, we outline the historical background and some of the recent advances in the field of three-dimensional organoids. We also highlight some of the current limitations of these systems and discuss potential avenues to further benefit biological research using three-dimensional modelling technologies.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.
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Affiliation(s)
- Sharmin Alhaque
- Scottish Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.,Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, Middlesex, UK
| | - Michael Themis
- Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, Middlesex, UK
| | - Hassan Rashidi
- Scottish Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK
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Bechmann N, Poser I, Seifert V, Greunke C, Ullrich M, Qin N, Walch A, Peitzsch M, Robledo M, Pacak K, Pietzsch J, Richter S, Eisenhofer G. Impact of Extrinsic and Intrinsic Hypoxia on Catecholamine Biosynthesis in Absence or Presence of Hif2α in Pheochromocytoma Cells. Cancers (Basel) 2019; 11:cancers11050594. [PMID: 31035382 PMCID: PMC6562431 DOI: 10.3390/cancers11050594] [Citation(s) in RCA: 31] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Revised: 04/24/2019] [Accepted: 04/25/2019] [Indexed: 01/10/2023] Open
Abstract
Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2α (MPC and MTT) or expressing both Hif1α and Hif2α (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hif1α- and Hif2α-driven effects we expressed Hif2α in MTT and MPC-mCherry cells (naturally lacking Hif2α). Presence of Hif2α resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1α in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2α under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24–48 h) by HIF1α. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2α-mediated phosphorylation of TH (permanent status).
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Affiliation(s)
- Nicole Bechmann
- Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden, Germany.
| | - Isabel Poser
- Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden, Germany.
| | - Verena Seifert
- Department of Radiopharmaceutical and Chemical Biology, Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Bautzner Landstrasse 400, 01328 Dresden, Germany.
| | - Christian Greunke
- Research Unit Analytical Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany; Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.
| | - Martin Ullrich
- Department of Radiopharmaceutical and Chemical Biology, Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Bautzner Landstrasse 400, 01328 Dresden, Germany.
| | - Nan Qin
- Division of Pediatric Neuro-Oncogenomics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
- German Consortium for Translational Cancer Research (DKTK), partner site Essen/Düsseldorf, 45147 Düsseldorf, Germany.
- Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Düsseldorf, 40225 Düsseldorf, Germany.
- Department of Neuropathology, Medical Faculty, Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany.
| | - Axel Walch
- Research Unit Analytical Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany; Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.
| | - Mirko Peitzsch
- Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden, Germany.
| | - Mercedes Robledo
- Hereditary Endocrine Cancer Group, CNIO, Madrid, Spain and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28029 Madrid, Spain.
| | - Karel Pacak
- Section on Medical Neuroendocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
| | - Jens Pietzsch
- Department of Radiopharmaceutical and Chemical Biology, Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Bautzner Landstrasse 400, 01328 Dresden, Germany.
- Department of Chemistry and Food Chemistry, School of Science, Technische Universität Dresden, Mommsenstrasse 9, 01062 Dresden, Germany.
| | - Susan Richter
- Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden, Germany.
| | - Graeme Eisenhofer
- Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden, Germany.
- Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden, Germany.
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Aktar R, Dietrich A, Tillner F, Kotb S, Löck S, Willers H, Baumann M, Krause M, Bütof R. Pre-clinical imaging for establishment and comparison of orthotopic non-small cell lung carcinoma: in search for models reflecting clinical scenarios. Br J Radiol 2019; 92:20180539. [PMID: 30215546 PMCID: PMC6541193 DOI: 10.1259/bjr.20180539] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2018] [Revised: 09/04/2018] [Accepted: 09/09/2018] [Indexed: 11/05/2022] Open
Abstract
OBJECTIVE: Clinically relevant animal models of non-small cell lung carcinoma (NSCLC) are required for the validation of novel treatments. We compared two different orthotopic transplantation techniques as well as imaging modalities to identify suitable mouse models mimicking clinical scenarios. METHODS: We used three genomically diverse NSCLC cell lines [National Cancer Institute (NCI)-H1703 adenosquamous cell carcinoma, NCI-H23 adenocarcinoma and A549 adenocarcinoma) for implanting tumour cells either as spheroids or cell suspension into lung parenchyma. Bioluminescence imaging (BLI) and contrast-enhanced cone beam CT (CBCT) were performed twice weekly to monitor tumour growth. Tumour histological data and microenvironmental parameters were determined. RESULTS: Tumour development after spheroid-based transplantation differs probably due to the integrity of spheroids, as H1703 developed single localised nodules, whereas H23 showed diffuse metastatic spread starting early after transplantation. A549 transplantation as cell suspension with the help of a stereotactic system was associated with initial single localised tumour growth and eventual metastatic spread. Imaging techniques were successfully applied to monitor longitudinal tumour growth: BLI revealed highly sensitive qualitative data, whereas CBCT was associated with less sensitive quantitative data. Histology revealed significant model-dependent heterogeneity in proliferation, hypoxia, perfusion and necrosis. CONCLUSION: Our developed orthotopic NSCLC tumours have similarity with biological growth behaviour comparable to that seen in the clinic and could therefore be used as attractive models to study tumour biology and evaluate new therapeutic strategies. The use of human cancer cell lines facilitates testing of different genomic tumour profiles that may affect treatment outcomes. ADVANCES IN KNOWLEDGE: The combination of different imaging modalities to identify tumour growth with subsequent use in treatment planning and orthotopic transplantation techniques to develop initially single lesions to ultimate metastases pave the way towards representative pre-clinical NSCLC models for experimental testing of novel therapeutic options in future studies.
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Affiliation(s)
| | | | | | | | | | - Henning Willers
- Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
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Pereira SGT, Hudoklin S, Kreft ME, Kostevsek N, Stuart MCA, Al-Jamal WT. Intracellular Activation of a Prostate Specific Antigen-Cleavable Doxorubicin Prodrug: A Key Feature Toward Prodrug-Nanomedicine Design. Mol Pharm 2019; 16:1573-1585. [DOI: 10.1021/acs.molpharmaceut.8b01257] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Affiliation(s)
- Sara G. T. Pereira
- School of Pharmacy, Queen’s University Belfast, 97 Lisburn Rd, Belfast BT9 7BL, U.K
| | - Samo Hudoklin
- University of Ljubljana, Faculty of Medicine, Institute of Cell Biology, Ljubljana, Slovenia
| | - Mateja Erdani Kreft
- University of Ljubljana, Faculty of Medicine, Institute of Cell Biology, Ljubljana, Slovenia
| | - Nina Kostevsek
- Department for Nanostructured Materials, Jozef Stefan Institute, Ljubljana, Slovenia
| | - Marc C. A. Stuart
- Electron Microscopy, University of Groningen, Nijenborgh 7, 9747AG Groningen, The Netherlands
| | - Wafa T. Al-Jamal
- School of Pharmacy, Queen’s University Belfast, 97 Lisburn Rd, Belfast BT9 7BL, U.K
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Smyrek I, Mathew B, Fischer SC, Lissek SM, Becker S, Stelzer EHK. E-cadherin, actin, microtubules and FAK dominate different spheroid formation phases and important elements of tissue integrity. Biol Open 2019; 8:bio.037051. [PMID: 30578251 PMCID: PMC6361217 DOI: 10.1242/bio.037051] [Citation(s) in RCA: 39] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Spheroids resemble features of tissues and serve as model systems to study cell–cell and cell–ECM interactions in non-adhesive three-dimensional environments. Although it is generally accepted that mature spheroids resemble tissue properties very well, no studies relate different phases in the spheroid formation processes that contribute to tissue integrity. Tissue integrity involves the cellular processes adhesion formation, adhesion reinforcement, rearrangement as well as proliferation. They maintain the structure and function of tissues and, upon dysregulation, contribute to malignancy. We investigated spheroid formation dynamics in cell lines of different metastatic potential. We dissected spheroid formation into phases of aggregation, compaction and growth to identify the respective contributions of E-cadherin, actin, microtubules and FAK. E-cadherin, actin and microtubules drive the first two phases. Microtubules and FAK are involved in the proliferation phase. FAK activity correlates with the metastatic potential of the cells. A robust computational model based on a very large number of experiments reveals the temporal resolution of cell adhesion. Our results provide novel hypotheses to unveil the general mechanisms that contribute to tissue integrity. Summary: The phases of spheroid formation resemble different stages of cell contact formation. This facilitates studying the temporal contribution of molecules in this process.
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Affiliation(s)
- I Smyrek
- Physical Biology/Physikalische Biologie (IZN, FB 15), Buchmann Institute for Molecular Life Sciences (BMLS), Cluster of Excellence Frankfurt - Macromolecular Complexes (CEF - MC), Goethe Universität - Frankfurt am Main (Campus Riedberg), Max-von-Laue-Straße 15, D-60348 Frankfurt am Main, Germany
| | - B Mathew
- Physical Biology/Physikalische Biologie (IZN, FB 15), Buchmann Institute for Molecular Life Sciences (BMLS), Cluster of Excellence Frankfurt - Macromolecular Complexes (CEF - MC), Goethe Universität - Frankfurt am Main (Campus Riedberg), Max-von-Laue-Straße 15, D-60348 Frankfurt am Main, Germany
| | - S C Fischer
- Physical Biology/Physikalische Biologie (IZN, FB 15), Buchmann Institute for Molecular Life Sciences (BMLS), Cluster of Excellence Frankfurt - Macromolecular Complexes (CEF - MC), Goethe Universität - Frankfurt am Main (Campus Riedberg), Max-von-Laue-Straße 15, D-60348 Frankfurt am Main, Germany
| | - S M Lissek
- Physical Biology/Physikalische Biologie (IZN, FB 15), Buchmann Institute for Molecular Life Sciences (BMLS), Cluster of Excellence Frankfurt - Macromolecular Complexes (CEF - MC), Goethe Universität - Frankfurt am Main (Campus Riedberg), Max-von-Laue-Straße 15, D-60348 Frankfurt am Main, Germany
| | - S Becker
- Physical Biology/Physikalische Biologie (IZN, FB 15), Buchmann Institute for Molecular Life Sciences (BMLS), Cluster of Excellence Frankfurt - Macromolecular Complexes (CEF - MC), Goethe Universität - Frankfurt am Main (Campus Riedberg), Max-von-Laue-Straße 15, D-60348 Frankfurt am Main, Germany
| | - E H K Stelzer
- Physical Biology/Physikalische Biologie (IZN, FB 15), Buchmann Institute for Molecular Life Sciences (BMLS), Cluster of Excellence Frankfurt - Macromolecular Complexes (CEF - MC), Goethe Universität - Frankfurt am Main (Campus Riedberg), Max-von-Laue-Straße 15, D-60348 Frankfurt am Main, Germany
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Rodríguez ME, Rizzi M, Caeiro L, Masip Y, Sánchez DO, Tekiel V. Transmigration of Trypanosoma cruzi Trypomastigotes through 3D Spheroids Mimicking Host Tissues. Methods Mol Biol 2019; 1955:165-177. [PMID: 30868526 DOI: 10.1007/978-1-4939-9148-8_12] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
While cellular invasion by T. cruzi trypomastigotes and intracellular amastigote replication are well-characterized events that have been described by using 2D monolayer cultures, other relevant parasite-host interactions, like the dynamics of tissue invasiveness, cannot be captured using monolayer cultures. Spheroids constitute a valuable three-dimensional (3D) culture system because they mimic the microarchitecture of tissues and provide an environment similar to the encountered in natural infections, which includes the presence of extracellular matrix as well as 3D cell-cell interactions. In this work, we describe a protocol for studying transmigration of T. cruzi trypomastigotes into 3D spheroids. In the experimental setup, cells and parasites are labelled with two fluorescent dyes, allowing their visualization by confocal microscopy. We also describe the general procedure and setting of the confocal microscope and downstream applications for acquisition and reconstruction of 3D images. This model was employed to analyze the transmigration of trypomastigotes from the highly virulent and pantropic RA T. cruzi strain. Of course, other aspects encountered by T. cruzi in the mammalian host environment can be studied with this methodology.
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Affiliation(s)
- Matías Exequiel Rodríguez
- Instituto de Investigaciones Biotecnológicas "Dr R. Ugalde", IIBIO, Universidad Nacional de San Martín (UNSAM) - CONICET, Pcia de Buenos Aires, Argentina
| | - Mariana Rizzi
- Instituto de Investigaciones Biotecnológicas "Dr R. Ugalde", IIBIO, Universidad Nacional de San Martín (UNSAM) - CONICET, Pcia de Buenos Aires, Argentina
| | - Lucas Caeiro
- Instituto de Investigaciones Biotecnológicas "Dr R. Ugalde", IIBIO, Universidad Nacional de San Martín (UNSAM) - CONICET, Pcia de Buenos Aires, Argentina
| | - Yamil Masip
- Instituto de Investigaciones Biotecnológicas "Dr R. Ugalde", IIBIO, Universidad Nacional de San Martín (UNSAM) - CONICET, Pcia de Buenos Aires, Argentina
| | - Daniel O Sánchez
- Instituto de Investigaciones Biotecnológicas "Dr R. Ugalde", IIBIO, Universidad Nacional de San Martín (UNSAM) - CONICET, Pcia de Buenos Aires, Argentina
| | - Valeria Tekiel
- Instituto de Investigaciones Biotecnológicas "Dr R. Ugalde", IIBIO, Universidad Nacional de San Martín (UNSAM) - CONICET, Pcia de Buenos Aires, Argentina.
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The micromass formation potential of human adipose-derived stromal cells isolated from different various origins. Head Face Med 2018; 14:19. [PMID: 30257689 PMCID: PMC6158821 DOI: 10.1186/s13005-018-0178-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2018] [Accepted: 09/20/2018] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Adult stem cells appear to be a promising subject for tissue engineering, representing an individual material for regeneration of aged and damaged cells. Especially adipose derived stromal cells (ADSC), which are easily to achieve, allow an encouraging perspective due to their capability of differentiating into miscellaneous cell types. Here we describe the in vitro formation of human subcutaneous, visceral and omental ADSC micromasses and compare their histological attributes while being cultivated on collagen membranes. METHODS Subcutaneous, visceral and omental fat tissue derived cells were isolated and processed according to standard protocols. Positively stained cells for CD13, CD44 and CD90 were cultivated on agarose in order to study micromass formation using a special method of cell tracking. Stained paraffin-embedded micromasses were analysed morphologically before and after being plated on collagen membranes. RESULTS The micromass formation process was similar in all three tissue types. Subcutaneous fat tissue derived micromasses turned out to develop a more homogeneous and compact shape than visceral and omental tissue. Nevertheless all micromasses adhered to collagen membranes with visible spreading of cells. The immune histochemical (IHC) staining of subcutaneous, visceral and omental ADSC micromasses shows a constant expression of CD13 and a decrease of CD44 and CD 90 expression within 28 days. After that period, omental fat cells don't show any expression of CD44. CONCLUSION In conclusion micromass formation and cultivation of all analysed fat tissues can be achieved, subcutaneous cells appearing to be the best material for regenerative concepts.
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Lazzari G, Nicolas V, Matsusaki M, Akashi M, Couvreur P, Mura S. Multicellular spheroid based on a triple co-culture: A novel 3D model to mimic pancreatic tumor complexity. Acta Biomater 2018; 78:296-307. [PMID: 30099198 DOI: 10.1016/j.actbio.2018.08.008] [Citation(s) in RCA: 167] [Impact Index Per Article: 23.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2018] [Revised: 07/11/2018] [Accepted: 08/08/2018] [Indexed: 12/12/2022]
Abstract
The preclinical drug screening of pancreatic cancer treatments suffers from the absence of appropriate models capable to reproduce in vitro the heterogeneous tumor microenvironment and its stiff desmoplasia. Driven by this pressing need, we describe in this paper the conception and the characterization of a novel 3D tumor model consisting of a triple co-culture of pancreatic cancer cells (PANC-1), fibroblasts (MRC-5) and endothelial cells (HUVEC), which assembled to form a hetero-type multicellular tumor spheroid (MCTS). By histological analyses and Selective Plain Illumination Microscopy (SPIM) we have monitored the spatial distribution of each cell type and the evolution of the spheroid composition. Results revealed the presence of a core rich in fibroblasts and fibronectin in which endothelial cells were homogeneously distributed. The integration of the three cell types enabled to reproduce in vitro with fidelity the influence of the surrounding environment on the sensitivity of cancer cells to chemotherapy. To our knowledge, this is the first time that a scaffold-free pancreatic cancer spheroid model combining both tumor and multiple stromal components has been designed. It holds the possibility to become an advantageous tool for a pertinent assessment of the efficacy of various therapeutic strategies. STATEMENT OF SIGNIFICANCE Pancreatic tumor microenvironment is characterized by abundant fibrosis and aberrant vasculature. Aiming to reproduce in vitro these features, cancer cells have been already co-cultured with fibroblasts or endothelial cells separately but the integration of both these essential components of the pancreatic tumor microenvironment in a unique system, although urgently needed, was still missing. In this study, we successfully integrated cellular and acellular microenvironment components (i.e., fibroblasts, endothelial cells, fibronectin) in a hetero-type scaffold-free multicellular tumor spheroid. This new 3D triple co-culture model closely mimicked the resistance to treatments observed in vivo, resulting in a reduction of cancer cell sensitivity to the anticancer treatment.
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Facile Tumor Spheroids Formation in Large Quantity with Controllable Size and High Uniformity. Sci Rep 2018; 8:6837. [PMID: 29717201 PMCID: PMC5931581 DOI: 10.1038/s41598-018-25203-3] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2018] [Accepted: 04/11/2018] [Indexed: 12/16/2022] Open
Abstract
A facile method for generation of tumor spheroids in large quantity with controllable size and high uniformity is presented. HCT-116 cells are used as a model cell line. Individual tumor cells are sparsely seeded onto petri-dishes. After a few days of growth, separated cellular islets are formed and then detached by dispase while maintaining their sheet shape. These detached cell sheets are transferred to dispase-doped media under orbital shaking conditions. Assisted by the shear flow under shaking and inhibition of cell-to-extracellular matrix junctions by dispase, the cell sheets curl up and eventually tumor spheroids are formed. The average size of the spheroids can be controlled by tuning the cell sheet culturing period and spheroid shaking period. The uniformity can be controlled by a set of sieves which were home-made using stainless steel meshes. Since this method is based on simple petri-dish cell culturing and shaking, it is rather facile for forming tumor spheroids with no theoretical quantity limit. This method has been used to form HeLa, A431 and U87 MG tumor spheroids and application of the formed tumor spheroids in drug screening is also demonstrated. The viability, 3D structure, and necrosis of the spheroids are characterized.
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Soranzo C, Della Torre G, Ingrosso A. Formation, Growth and Morphology of Multicellular Tumor Spheroids from a Human Colon Carcinoma Cell Line (LoVo). TUMORI JOURNAL 2018; 72:459-67. [PMID: 3798565 DOI: 10.1177/030089168607200502] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
LoVo human colon carcinoma cells cultured by a liquid overlay technique form and grow as multicellular tumor spheroids. The growth curve of LoVo spheroids exhibits Gompertzian growth kinetics, i.e., exponential growth for 10 days, followed by exponential retardation in the rate of growth. Doubling time in the exponential growth phase is longer than in monolayer cultures (5 days for LoVo spheroids vs. 37 h for monolayers). When LoVo spheroids reach a diameter of about 300 ūm, a necrotic core appears in their center and continuously increases with spheroid growth. The cell ultrastructure and organization in spheroids closely resemble those of the same cells when grown as tumors in vivo or as monolayer, i.e. intestinal epithelium, desmosomes, intracytoplasmic lumina and acinar structures. Individual cells from spheroids can be obtained by trypsinization and assayed for colony formation. LoVo spheroids provide a model which can be readily manipulated and appears to be suitable for evaluation of anticancer drugs. A comparison of LoVo spheroids exposed to doxorubicin with the same cells grown in monolayers emphasized the role of cell organization in determining drug resistance.
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Almela T, Al-Sahaf S, Brook IM, Khoshroo K, Rasoulianboroujeni M, Fahimipour F, Tahriri M, Dashtimoghadam E, Bolt R, Tayebi L, Moharamzadeh K. 3D printed tissue engineered model for bone invasion of oral cancer. Tissue Cell 2018; 52:71-77. [PMID: 29857831 DOI: 10.1016/j.tice.2018.03.009] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2018] [Revised: 02/09/2018] [Accepted: 03/19/2018] [Indexed: 12/16/2022]
Abstract
Recent advances in three-dimensional printing technology have led to a rapid expansion of its applications in tissue engineering. The present study was designed to develop and characterize an in vitro multi-layered human alveolar bone, based on a 3D printed scaffold, combined with tissue engineered oral mucosal model. The objective was to incorporate oral squamous cell carcinoma (OSCC) cell line spheroids to the 3D model at different anatomical levels to represent different stages of oral cancer. Histological evaluation of the 3D tissue model revealed a tri-layered structure consisting of distinct epithelial, connective tissue, and bone layers; replicating normal oral tissue architecture. The mucosal part showed a well-differentiated stratified oral squamous epithelium similar to that of the native tissue counterpart, as demonstrated by immunohistochemistry for cytokeratin 13 and 14. Histological assessment of the cancerous models demonstrated OSCC spheroids at three depths including supra-epithelial level, sub-epithelial level, and deep in the connective tissue-bone interface. The 3D tissue engineered composite model closely simulated the native oral hard and soft tissues and has the potential to be used as a valuable in vitro model for the investigation of bone invasion of oral cancer and for the evaluation of novel diagnostic or therapeutic approaches to manage OSCC in the future.
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Affiliation(s)
- Thafar Almela
- School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield, S10 2TA, UK.
| | - Sarmad Al-Sahaf
- School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield, S10 2TA, UK
| | - Ian M Brook
- School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield, S10 2TA, UK
| | - Kimia Khoshroo
- Department of Developmental Sciences, School of Dentistry, Marquette University, Milwaukee, WI, 53233, USA
| | - Morteza Rasoulianboroujeni
- Department of Developmental Sciences, School of Dentistry, Marquette University, Milwaukee, WI, 53233, USA
| | - Farahnaz Fahimipour
- Department of Developmental Sciences, School of Dentistry, Marquette University, Milwaukee, WI, 53233, USA
| | - Mohammadreza Tahriri
- Department of Developmental Sciences, School of Dentistry, Marquette University, Milwaukee, WI, 53233, USA
| | - Erfan Dashtimoghadam
- Department of Developmental Sciences, School of Dentistry, Marquette University, Milwaukee, WI, 53233, USA
| | - Robert Bolt
- School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield, S10 2TA, UK
| | - Lobat Tayebi
- Department of Developmental Sciences, School of Dentistry, Marquette University, Milwaukee, WI, 53233, USA; Biomaterials and Advanced Drug Delivery Laboratory, School of Medicine, Stanford University, Palo Alto, CA, USA; Department of Engineering Science, University of Oxford, Oxford, OX1 3PJ, UK
| | - Keyvan Moharamzadeh
- School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield, S10 2TA, UK; Department of Developmental Sciences, School of Dentistry, Marquette University, Milwaukee, WI, 53233, USA
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Schäfer S, Urban K, Gerber M, Dekiff M, Dirksen D, Plate U. Dynamic behavior of different quantities of osteoblasts during formation of micromass cultures. Cytometry A 2018; 93:458-463. [PMID: 29493890 DOI: 10.1002/cyto.a.23347] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2017] [Revised: 01/18/2018] [Accepted: 02/04/2018] [Indexed: 11/07/2022]
Abstract
Implantation of micromass cultures of osteoblastic cells offers the possibility of scaffold free tissue engineering for example, regeneration of bone defects. However, the details of cell dynamics during the formation of these micromasses are still not well understood. This study aims to investigate and clarify the extent to which cell quantity influences the dynamics of micromass formation of osteoblastic cell cultures. For this purpose, the migration and aggregation during this process are investigated by optical inspection employing image processing software that allows for automated tracking of cell groups using digital image correlation. An exponential time behavior is observed with respect to the velocity of the cells and the distance of the cells to their common center of gravity. Characteristic time constants are derived as quantitative measures of the cell dynamics. The results indicate that the time constants strongly depend on the quantity of cells, that is, will decrease with increasing cell quantity. © 2018 International Society for Advancement of Cytometry.
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Affiliation(s)
- Susanne Schäfer
- Department of Maxillofacial Surgery, University of Muenster, Waldeyer Str. 30, Muenster, D-48149, Germany
| | - Kent Urban
- Department of Periodontology and Restorative Dentistry, University of Muenster, Waldeyer Str. 30, Muenster, D-48149, Germany
| | - Maria Gerber
- Department of Maxillofacial Surgery, University of Muenster, Waldeyer Str. 30, Muenster, D-48149, Germany
| | - Markus Dekiff
- Department of Prosthetic Dentistry and Biomaterials, University of Muenster, Waldeyer Str. 30, Muenster, D-48149, Germany
| | - Dieter Dirksen
- Department of Prosthetic Dentistry and Biomaterials, University of Muenster, Waldeyer Str. 30, Muenster, D-48149, Germany
| | - Ulrich Plate
- Department of Maxillofacial Surgery, University of Muenster, Waldeyer Str. 30, Muenster, D-48149, Germany
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Thomsen AR, Aldrian C, Bronsert P, Thomann Y, Nanko N, Melin N, Rücker G, Follo M, Grosu AL, Niedermann G, Layer PG, Heselich A, Lund PG. A deep conical agarose microwell array for adhesion independent three-dimensional cell culture and dynamic volume measurement. LAB ON A CHIP 2017; 18:179-189. [PMID: 29211089 DOI: 10.1039/c7lc00832e] [Citation(s) in RCA: 53] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Multicellular spheroids represent a well-established 3D model to study healthy and diseased cells in vitro. The use of conventional 3D cell culture platforms for the generation of multicellular spheroids is limited to cell types that easily self-assemble into spheroids because less adhesive cells fail to form stable aggregates. A high-precision micromoulding technique developed in our laboratory produces deep conical agarose microwell arrays that allow the cultivation of uniform multicellular aggregates, irrespective of the spheroid formation capacity of the cells. Such hydrogel arrays warrant a steady nutrient supply for several weeks, permit live volumetric measurements to monitor cell growth, enable immunohistochemical staining, fluorescence-based microscopy, and facilitate immediate harvesting of cell aggregates. This system also allows co-cultures of two distinct cell types either in direct cell-cell contact or at a distance as the hydrogel permits diffusion of soluble compounds. Notably, we show that co-culture of a breast cancer cell line with bone marrow stromal cells enhances 3D growth of the cancer cells in this system.
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Affiliation(s)
- Andreas R Thomsen
- Department of Radiation Oncology, Medical Center - University of Freiburg, Germany.
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Raza A, Colley HE, Baggaley E, Sazanovich IV, Green NH, Weinstein JA, Botchway SW, MacNeil S, Haycock JW. Oxygen Mapping of Melanoma Spheroids using Small Molecule Platinum Probe and Phosphorescence Lifetime Imaging Microscopy. Sci Rep 2017; 7:10743. [PMID: 28878302 PMCID: PMC5587740 DOI: 10.1038/s41598-017-11153-9] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2017] [Accepted: 08/21/2017] [Indexed: 02/05/2023] Open
Abstract
Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new diagnostic tools to determine and exploit these variations. Oxygen is an efficient quencher of emission of many phosphorescent compounds, thus oxygen concentration could in many cases be derived directly from relative emission intensity and lifetime. In this study, we extend our previous work on phosphorescent, low molecular weight platinum(II) complex as an oxygen sensing probe to study the variation in oxygen concentration in a viable multicellular 3D human tumour model. The data shows one of the first examples of non-invasive, real-time oxygen mapping across a melanoma tumour spheroid using one-photon phosphorescence lifetime imaging microscopy (PLIM) and a small molecule oxygen sensitive probe. These measurements were quantitative and enabled real time oxygen mapping with high spatial resolution. This combination presents as a valuable tool for optical detection of both physiological and pathological oxygen levels in a live tissue mass and we suggest has the potential for broader clinical application.
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Affiliation(s)
- Ahtasham Raza
- Materials Science & Engineering, University of Sheffield, Sheffield, S3 7HQ, UK
| | - Helen E Colley
- School of Clinical Dentistry, University of Sheffield, Sheffield, S10 2TA, UK
| | | | - Igor V Sazanovich
- Research Complex at Harwell (CLF), STFC Rutherford Appleton Laboratory, Oxford, OX11 0QX, UK
| | - Nicola H Green
- Materials Science & Engineering, University of Sheffield, Sheffield, S3 7HQ, UK
| | - Julia A Weinstein
- Department of Chemistry, University of Sheffield, Sheffield, S3 7HF, UK
| | - Stanley W Botchway
- Research Complex at Harwell (CLF), STFC Rutherford Appleton Laboratory, Oxford, OX11 0QX, UK
| | - Sheila MacNeil
- Materials Science & Engineering, University of Sheffield, Sheffield, S3 7HQ, UK
| | - John W Haycock
- Materials Science & Engineering, University of Sheffield, Sheffield, S3 7HQ, UK.
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Blumlein A, Williams N, McManus JJ. The mechanical properties of individual cell spheroids. Sci Rep 2017; 7:7346. [PMID: 28779182 PMCID: PMC5544704 DOI: 10.1038/s41598-017-07813-5] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2016] [Accepted: 07/03/2017] [Indexed: 12/18/2022] Open
Abstract
The overall physical properties of tissues emerge in a complex manner from the properties of the component cells and other constituent materials from which the tissue is formed, across multiple length scales ranging from nanometres to millimetres. Recent studies have suggested that interfacial tension between cells contributes significantly to the mechanical properties of tissues and that the overall surface tension is determined by the ratio of adhesion tension to cortical tension. Using cavitation rheology (CR), we have measured the interfacial properties and the elastic modulus of spheroids formed from HEK cells. By comparing the work of bubble formation with deformation of the cell spheroid at different length scales, we have estimated the cortical tension for HEK cells. This innovative approach to understanding the fundamental physical properties associated with tissue mechanics may guide new approaches for the generation of materials to replace or regenerate damaged or diseased tissues.
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Affiliation(s)
- Alice Blumlein
- Department of Chemistry, Maynooth University, Maynooth, Co. Kildare, Ireland
| | - Noel Williams
- Department of Chemistry, Maynooth University, Maynooth, Co. Kildare, Ireland
| | - Jennifer J McManus
- Department of Chemistry, Maynooth University, Maynooth, Co. Kildare, Ireland.
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Kaistha BP, Krattenmacher A, Fredebohm J, Schmidt H, Behrens D, Widder M, Hackert T, Strobel O, Hoheisel JD, Gress TM, Buchholz M. The deubiquitinating enzyme USP5 promotes pancreatic cancer via modulating cell cycle regulators. Oncotarget 2017; 8:66215-66225. [PMID: 29029505 PMCID: PMC5630405 DOI: 10.18632/oncotarget.19882] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2017] [Accepted: 07/11/2017] [Indexed: 11/25/2022] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid tumors. With an overall five-year survival rate remaining below 6%, there is an explicit need to search for new molecular targets for therapeutic interventions. We undertook a barcode labelled short-hairpin (shRNA) library screen in pancreatic cancer cells in order to identify novel genes promoting cancer survival and progression. Among the candidate genes identified in this screen was the deubiquitinase USP5, which subsequent gene expression analyses demonstrated to be significantly upregulated in primary human pancreatic cancer tissues. Using different knockdown approaches, we show that expression of USP5 is essential for the proliferation and survival of pancreatic cancer cells, tested under different 2D and 3D cell culture conditions as well as in in vivo experiments. These growth inhibition effects upon knockdown of USP5 are mediated primarily by the attenuation of G1/S phase transition in the cells, which is accompanied by accumulation of DNA damage, upregulation of p27, and increased apoptosis rates. Since USP5 is overexpressed in cancer tissues, it can thus potentially serve as a new target for therapeutic interventions, especially given the fact that deubiquitinases are currently emerging as new class of attractive drug targets in cancer.
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Affiliation(s)
- Brajesh P Kaistha
- Department of Medicine, Division of Gastroenterology, Endocrinology and Metabolism, Philipps-University Marburg, Marburg, Germany
| | - Anja Krattenmacher
- Department of Medicine, Division of Gastroenterology, Endocrinology and Metabolism, Philipps-University Marburg, Marburg, Germany
| | - Johannes Fredebohm
- Division of Functional Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Harald Schmidt
- Department of Medicine, Division of Gastroenterology, Endocrinology and Metabolism, Philipps-University Marburg, Marburg, Germany
| | - Diana Behrens
- Experimantal Pharmacology and Oncology (EPO Berlin-Buch), Berlin, Germany
| | - Miriam Widder
- Institute for Bioprocessing and Analytical Measurement Techniques (IBA-Heiligenstadt), Heilbad Heiligenstadt, Germany
| | - Thilo Hackert
- Department of Surgery, University Clinic Heidelberg, Heidelberg, Germany
| | - Oliver Strobel
- Department of Surgery, University Clinic Heidelberg, Heidelberg, Germany
| | - Jörg D Hoheisel
- Division of Functional Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Thomas M Gress
- Department of Medicine, Division of Gastroenterology, Endocrinology and Metabolism, Philipps-University Marburg, Marburg, Germany
| | - Malte Buchholz
- Department of Medicine, Division of Gastroenterology, Endocrinology and Metabolism, Philipps-University Marburg, Marburg, Germany
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