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1
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McGeachan RI, Meftah S, Taylor LW, Catterson JH, Negro D, Bonthron C, Holt K, Tulloch J, Rose JL, Gobbo F, Chang YY, Elliott J, McLay L, King D, Liaquat I, Spires-Jones TL, Booker SA, Brennan PM, Durrant CS. Divergent actions of physiological and pathological amyloid-β on synapses in live human brain slice cultures. Nat Commun 2025; 16:3753. [PMID: 40307257 PMCID: PMC12044016 DOI: 10.1038/s41467-025-58879-z] [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: 02/27/2024] [Accepted: 04/01/2025] [Indexed: 05/02/2025] Open
Abstract
In Alzheimer's disease, amyloid beta (Aβ) and tau pathology are thought to drive synapse loss. However, there is limited information on how endogenous levels of tau, Aβ and other biomarkers relate to patient characteristics, or how manipulating physiological levels of Aβ impacts synapses in living adult human brain. Using live human brain slice cultures, we report that Aβ1-40 and tau release levels vary with donor age and brain region, respectively. Release of other biomarkers such as KLK-6, NCAM-1, and Neurogranin vary between brain region, while TDP-43 and NCAM-1 release is impacted by sex. Pharmacological manipulation of Aβ in either direction results in a loss of synaptophysin puncta, with increased physiological Aβ triggering potentially compensatory synaptic transcript changes. In contrast, treatment with Aβ-containing Alzheimer's disease brain extract results in post-synaptic Aβ uptake and pre-synaptic puncta loss without affecting synaptic transcripts. These data reveal distinct effects of physiological and pathological Aβ on synapses in human brain tissue.
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Affiliation(s)
- Robert I McGeachan
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
- The Hospital for Small Animals, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Edinburgh, UK
| | - Soraya Meftah
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Lewis W Taylor
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - James H Catterson
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Danilo Negro
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Calum Bonthron
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Kristján Holt
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Jane Tulloch
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Jamie L Rose
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Francesco Gobbo
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Ya Yin Chang
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Jamie Elliott
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Lauren McLay
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Declan King
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Imran Liaquat
- Department of Clinical Neuroscience, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh, UK
| | - Tara L Spires-Jones
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK
| | - Sam A Booker
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK
- Simons Initiative for the Developing Brain, The University of Edinburgh, Edinburgh, UK
| | - Paul M Brennan
- Department of Clinical Neuroscience, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh, UK
- Translational Neurosurgery, The Centre for Clinical Brain Sciences, The University of Edinburgh, Edinburgh, UK
- Cancer Research UK Brain Tumour Centre of Excellence, CRUK Edinburgh Centre, The University of Edinburgh, Edinburgh, UK
| | - Claire S Durrant
- Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK.
- UK Dementia Research Institute, The University of Edinburgh, Edinburgh, UK.
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2
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Bak A, Koch H, van Loo KMJ, Schmied K, Gittel B, Weber Y, Ort J, Schwarz N, Tauber SC, Wuttke TV, Delev D. Human organotypic brain slice cultures: a detailed and improved protocol for preparation and long-term maintenance. J Neurosci Methods 2024; 404:110055. [PMID: 38184112 DOI: 10.1016/j.jneumeth.2023.110055] [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: 09/30/2023] [Revised: 12/11/2023] [Accepted: 12/31/2023] [Indexed: 01/08/2024]
Abstract
The investigation of the human brain at cellular and microcircuit level remains challenging due to the fragile viability of neuronal tissue, inter- and intra-variability of the samples and limited availability of human brain material. Especially brain slices have proven to be an excellent source to investigate brain physiology and disease at cellular and small network level, overcoming the temporal limits of acute slices. Here we provide a revised, detailed protocol of the production and in-depth knowledge on long-term culturing of such human organotypic brain slice cultures for research purposes. We highlight the critical pitfalls of the culturing process of the human brain tissue and present exemplary results on viral expression, single-cell Patch-Clamp recordings, as well as multi-electrode array recordings as readouts for culture viability, enabling the use of organotypic brain slice cultures of these valuable tissue samples for basic neuroscience and disease modeling (Fig. 1).
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Affiliation(s)
- Aniella Bak
- Department of Epileptology, Neurology, RWTH Aachen University Hospital, Aachen, Germany.
| | - Henner Koch
- Department of Epileptology, Neurology, RWTH Aachen University Hospital, Aachen, Germany
| | - Karen M J van Loo
- Department of Epileptology, Neurology, RWTH Aachen University Hospital, Aachen, Germany; Department of Neurosurgery, RWTH Aachen University, Aachen, Germany
| | - Katharina Schmied
- Department of Epileptology, Neurology, RWTH Aachen University Hospital, Aachen, Germany
| | - Birgit Gittel
- Department of Epileptology, Neurology, RWTH Aachen University Hospital, Aachen, Germany
| | - Yvonne Weber
- Department of Epileptology, Neurology, RWTH Aachen University Hospital, Aachen, Germany
| | - Jonas Ort
- Department of Neurosurgery, RWTH Aachen University, Aachen, Germany
| | - Niklas Schwarz
- Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany
| | - Simone C Tauber
- Department of Neurology, RWTH Aachen University Hospital, Aachen, Germany
| | - Thomas V Wuttke
- Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany; Department of Neurosurgery, University of Tübingen, Tübingen, Germany
| | - Daniel Delev
- Department of Neurosurgery, RWTH Aachen University, Aachen, Germany; Neurosurgical Artificial Intelligence Laboratory Aachen (NAILA), RWTH Aachen University Hospital, Aachen, Germany; Department of Neurosurgery, University of Erlangen, Erlangen, Germany
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3
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Li Y, Wang J, Song SR, Lv SQ, Qin JH, Yu SC. Models for evaluating glioblastoma invasion along white matter tracts. Trends Biotechnol 2024; 42:293-309. [PMID: 37806896 DOI: 10.1016/j.tibtech.2023.09.005] [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: 05/17/2023] [Revised: 08/31/2023] [Accepted: 09/18/2023] [Indexed: 10/10/2023]
Abstract
White matter tracts (WMs) are one of the main invasion paths of glioblastoma multiforme (GBM). The lack of ideal research models hinders our understanding of the details and mechanisms of GBM invasion along WMs. To date, many potential in vitro models have been reported; nerve fiber culture models and nanomaterial models are biocompatible, and the former have electrically active neurons. Brain slice culture models, organoid models, and microfluidic chip models can simulate the real brain and tumor microenvironment (TME), which contains a variety of cell types. These models are closer to the real in vivo environment and are helpful for further studying not only invasion along WMs by GBM, but also perineural invasion and brain metastasis by solid tumors.
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Affiliation(s)
- Yao Li
- Department of Stem Cell and Regenerative Medicine, Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Chongqing 400038, China; International Joint Research Center for Precision Biotherapy, Ministry of Science and Technology, Chongqing 400038, China; Key Laboratory of Cancer Immunopathology, Ministry of Education, Chongqing 400038, China; Department of Neurosurgery, Xinqiao Hospital, Chongqing 400037, China
| | - Jun Wang
- Department of Stem Cell and Regenerative Medicine, Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Chongqing 400038, China; International Joint Research Center for Precision Biotherapy, Ministry of Science and Technology, Chongqing 400038, China; Key Laboratory of Cancer Immunopathology, Ministry of Education, Chongqing 400038, China; Jin-feng Laboratory, Chongqing 401329, China
| | - Si-Rong Song
- Department of Stem Cell and Regenerative Medicine, Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Chongqing 400038, China; International Joint Research Center for Precision Biotherapy, Ministry of Science and Technology, Chongqing 400038, China; Key Laboratory of Cancer Immunopathology, Ministry of Education, Chongqing 400038, China
| | - Sheng-Qing Lv
- Department of Neurosurgery, Xinqiao Hospital, Chongqing 400037, China
| | - Jian-Hua Qin
- Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Niaoning 116023, China.
| | - Shi-Cang Yu
- Department of Stem Cell and Regenerative Medicine, Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Chongqing 400038, China; International Joint Research Center for Precision Biotherapy, Ministry of Science and Technology, Chongqing 400038, China; Key Laboratory of Cancer Immunopathology, Ministry of Education, Chongqing 400038, China; Jin-feng Laboratory, Chongqing 401329, China.
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4
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Chong HK, Ma Z, Wong KKC, Morokoff A, French C. An In Vitro Brain Tumour Model in Organotypic Slice Cultures Displaying Epileptiform Activity. Brain Sci 2023; 13:1451. [PMID: 37891819 PMCID: PMC10605659 DOI: 10.3390/brainsci13101451] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2023] [Revised: 10/04/2023] [Accepted: 10/07/2023] [Indexed: 10/29/2023] Open
Abstract
Brain tumours have significant impacts on patients' quality of life, and current treatments have limited effectiveness. To improve understanding of tumour development and explore new therapies, researchers rely on experimental models. However, reproducing tumour-associated epilepsy (TAE) in these models has been challenging. Existing models vary from cell lines to in vivo studies, but in vivo models are resource-intensive and often fail to mimic crucial features like seizures. In this study, we developed a technique in which normal rat organotypic brain tissue is implanted with an aggressive brain tumour. This method produces a focal invasive lesion that preserves neural responsiveness and exhibits epileptiform hyperexcitability. It allows for real-time imaging of tumour growth and invasion for up to four weeks and microvolume fluid sampling analysis of different regions, including the tumour, brain parenchyma, and peritumoral areas. The tumour cells expand and infiltrate the organotypic slice, resembling in vivo behaviour. Spontaneous seizure-like events occur in the tumour slice preparation and can be induced with stimulation or high extracellular potassium. Furthermore, we assess extracellular fluid composition in various regions of interest. This technique enables live cell confocal microscopy to record real-time tumour invasion properties, whilst maintaining neural excitability, generating field potentials, and epileptiform discharges, and provides a versatile preparation for the study of major clinical problems of tumour-associated epilepsy.
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Affiliation(s)
- Harvey K. Chong
- Neural Dynamics Laboratory, Department of Medicine, University of Melbourne, Melbourne, VIC 3052, Australia; (H.K.C.); (K.K.C.W.); (A.M.); (C.F.)
| | - Ziang Ma
- Neural Dynamics Laboratory, Department of Medicine, University of Melbourne, Melbourne, VIC 3052, Australia; (H.K.C.); (K.K.C.W.); (A.M.); (C.F.)
| | - Kendrew Ka Chuon Wong
- Neural Dynamics Laboratory, Department of Medicine, University of Melbourne, Melbourne, VIC 3052, Australia; (H.K.C.); (K.K.C.W.); (A.M.); (C.F.)
| | - Andrew Morokoff
- Neural Dynamics Laboratory, Department of Medicine, University of Melbourne, Melbourne, VIC 3052, Australia; (H.K.C.); (K.K.C.W.); (A.M.); (C.F.)
- Department of Medicine, Royal Melbourne Hospital, Parkville, Melbourne, VIC 3000, Australia
| | - Chris French
- Neural Dynamics Laboratory, Department of Medicine, University of Melbourne, Melbourne, VIC 3052, Australia; (H.K.C.); (K.K.C.W.); (A.M.); (C.F.)
- Department of Medicine, Royal Melbourne Hospital, Parkville, Melbourne, VIC 3000, Australia
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5
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Decotret LR, Shi R, Thomas KN, Hsu M, Pallen CJ, Bennewith KL. Development and validation of an advanced ex vivo brain slice invasion assay to model glioblastoma cell invasion into the complex brain microenvironment. Front Oncol 2023; 13:976945. [PMID: 36793608 PMCID: PMC9923402 DOI: 10.3389/fonc.2023.976945] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Accepted: 01/04/2023] [Indexed: 01/31/2023] Open
Abstract
Organotypic cultures of murine brain slices are well-established tools in neuroscience research, including electrophysiology studies, modeling neurodegeneration, and cancer research. Here, we present an optimized ex vivo brain slice invasion assay that models glioblastoma multiforme (GBM) cell invasion into organotypic brain slices. Using this model, human GBM spheroids can be implanted with precision onto murine brain slices and cultured ex vivo to allow tumour cell invasion into the brain tissue. Traditional top-down confocal microscopy allows for imaging of GBM cell migration along the top of the brain slice, but there is limited resolution of tumour cell invasion into the slice. Our novel imaging and quantification technique involves embedding stained brain slices into an agar block, re-sectioning the slice in the Z-direction onto slides, and then using confocal microscopy to image cellular invasion into the brain tissue. This imaging technique allows for the visualization of invasive structures beneath the spheroid that would otherwise go undetected using traditional microscopy approaches. Our ImageJ macro (BraInZ) allows for the quantification of GBM brain slice invasion in the Z-direction. Importantly, we note striking differences in the modes of motility observed when GBM cells invade into Matrigel in vitro versus into brain tissue ex vivo highlighting the importance of incorporating the brain microenvironment when studying GBM invasion. In summary, our version of the ex vivo brain slice invasion assay improves upon previously published models by more clearly differentiating between migration along the top of the brain slice versus invasion into the slice.
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Affiliation(s)
- Lisa R Decotret
- Department of Integrative Oncology, BC Cancer, Vancouver, BC, Canada.,Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada
| | - Rocky Shi
- Department of Integrative Oncology, BC Cancer, Vancouver, BC, Canada.,Interdisciplinary Oncology Program, University of British Columbia, Vancouver, BC, Canada
| | - Kiersten N Thomas
- Department of Integrative Oncology, BC Cancer, Vancouver, BC, Canada.,Interdisciplinary Oncology Program, University of British Columbia, Vancouver, BC, Canada
| | - Manchi Hsu
- Department of Integrative Oncology, BC Cancer, Vancouver, BC, Canada
| | - Catherine J Pallen
- Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada.,Michael Cuccione Childhood Cancer Research Program, BC Children's Hospital Research Institute, Vancouver, BC, Canada.,Department of Pediatrics, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada
| | - Kevin L Bennewith
- Department of Integrative Oncology, BC Cancer, Vancouver, BC, Canada.,Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada.,Interdisciplinary Oncology Program, University of British Columbia, Vancouver, BC, Canada
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6
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Fabrication of innocuous hydrogel scaffolds based on modified dextran for biotissues. Carbohydr Res 2022; 522:108699. [DOI: 10.1016/j.carres.2022.108699] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2022] [Revised: 09/16/2022] [Accepted: 10/03/2022] [Indexed: 11/18/2022]
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7
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Sugawara T, Kobayashi D, Maehara T. Histopathological Investigation of Meningioma Capsule with Respect to Tumor Cell Invasion. Neurol Med Chir (Tokyo) 2022; 62:431-437. [PMID: 35944984 PMCID: PMC9534571 DOI: 10.2176/jns-nmc.2021-0402] [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] [Indexed: 11/20/2022] Open
Abstract
No previous study has histopathologically investigated whether a meningioma capsule presents with tumor cells. We investigated which types of tumor capsules (TCs) included tumor cells to help intraoperatively determine those TCs that do not need to be removed and have a low recurrence risk. We investigated 22 specimens of 14 newly diagnosed meningiomas from February 2011 to June 2021. The capsules were classified into three types: TC, capsule-like thickened arachnoid membrane (CAM), and extended membrane (EM). Capsule properties were scored by hardness (soft = 1, medium = 2, hard = 3) and transparency (high = 1, medium = 2, low = 3). The hardness, transparency, and score sums were compared between capsules with and without tumor invasion in the CAM and EM types. The mean follow-up duration was 40.6 months, and there was only one recurrence in a remote location from the residual capsule. Nine capsules were classified as TC, seven as CAM, and six as EM. The tumor cells invaded 88.9% of TCs, 42.9% of CAMs, and 50% of EMs. The hardness, transparency, and score sums for CAMs with tumor invasion were lower than those for CAMs without tumor invasion, although not significant (P = 0.114, P = 0.114, P = 0.057, respectively). A thickened TC or soft and highly transparent CAM indicated a high risk for tumor cell invasion; thus, such cases require a careful and long-term follow-up. Hard and low transparent residual CAMs may have had a low risk for tumor invasion; therefore, leaving such capsules that tightly adhere to the eloquent cortex can be theoretically justified to avoid damaging the brain surface.
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Singh M, Dahal A, Brastianos PK. Preclinical Solid Tumor Models to Study Novel Therapeutics in Brain Metastases. Curr Protoc 2021; 1:e284. [PMID: 34762346 PMCID: PMC8597918 DOI: 10.1002/cpz1.284] [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] [Indexed: 11/09/2022]
Abstract
Metastases are the most common malignancy of the adult central nervous system and are becoming an increasingly troubling problem in oncology largely due to the lack of successful therapeutic options. The limited selection of treatments is a result of the currently poor understanding of the biological mechanisms of metastatic development, which in turn is difficult to achieve because of limited preclinical models that can accurately represent the clinical progression of metastasis. Described in this article are in vitro and in vivo model systems that are used to enhance the understanding of metastasis and to identify new therapies for the treatment of brain metastasis. © 2021 Wiley Periodicals LLC.
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Affiliation(s)
- Mohini Singh
- Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA
| | - Ashish Dahal
- Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA
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9
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Tamura R, Miyoshi H, Morimoto Y, Oishi Y, Sampetrean O, Iwasawa C, Mine Y, Saya H, Yoshida K, Okano H, Toda M. Gene Therapy Using Neural Stem/Progenitor Cells Derived from Human Induced Pluripotent Stem Cells: Visualization of Migration and Bystander Killing Effect. Hum Gene Ther 2021; 31:352-366. [PMID: 32075424 DOI: 10.1089/hum.2019.326] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Glioblastoma is the most aggressive brain tumor characterized by diffuse infiltration into the normal brain parenchyma. Neural stem cells are known to possess the tumor-tropic migratory capacity and thus can be used as cellular vehicles for targeted delivery of therapeutic agents. In the present study, we evaluated the efficacy of herpes simplex virus thymidine kinase (HSV-TK) suicide gene therapy for glioblastoma using neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs). Although transduction of hiPSCs is preferable for a safe and stable supply in the clinical setting, high-level and/or constitutive HSV-TK expression was highly cytotoxic to hiPSCs. To overcome this problem, we used the tetracycline-inducible system to control the expression of HSV-TK. hiPSC-derived NS/PCs expressing HSV-TK were transplanted in an orthotopic xenograft mouse model of human glioblastoma. Glioblastoma cell growth in mice was dramatically inhibited following ganciclovir (GCV) administration. Survival of the mice was significantly prolonged with administration of GCV compared with control groups. Time-lapse imaging of organotypic brain slice cultures first demonstrated the directional migration of NS/PCs toward glioblastoma cells and the bystander killing effect upon GCV treatment. hiPSC-derived NS/PCs with HSV-TK/GCV suicide gene system may have considerable therapeutic potential for the treatment of glioblastoma. Color images are available online.
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Affiliation(s)
- Ryota Tamura
- Departments of Neurosurgery, Keio University School of Medicine, Tokyo, Japan
| | - Hiroyuki Miyoshi
- Departments of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Yukina Morimoto
- Departments of Neurosurgery, Keio University School of Medicine, Tokyo, Japan
| | - Yumiko Oishi
- Departments of Neurosurgery, Keio University School of Medicine, Tokyo, Japan
| | - Oltea Sampetrean
- Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan
| | - Chizuru Iwasawa
- Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo, Japan
| | - Yutaka Mine
- Departments of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Hideyuki Saya
- Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan
| | - Kazunari Yoshida
- Departments of Neurosurgery, Keio University School of Medicine, Tokyo, Japan
| | - Hideyuki Okano
- Departments of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Masahiro Toda
- Departments of Neurosurgery, Keio University School of Medicine, Tokyo, Japan
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Abstract
The physical microenvironment of cells plays a fundamental role in regulating cellular behavior and cell fate, especially in the context of cancer metastasis. For example, capillary deformation can destroy arrested circulating tumor cells while the dense extracellular matrix can form a physical barrier for invading cancer cells. Understanding how metastatic cancer cells overcome the challenges brought forth by physical confinement can help in developing better therapeutics that can put a stop to this migratory stage of the metastatic cascade. Numerous in vivo and in vitro assays have been developed to recapitulate the metastatic processes and study cancer cell migration in a confining microenvironment. In this review, we summarize some of the representative techniques and the exciting new findings. We critically review the advantages, as well as challenges associated with these tools and methodologies, and provide a guide on the applications that they are most suited for. We hope future efforts that push forward our current understanding on metastasis under confinement can lead to novel and more effective diagnostic and therapeutic strategies against this dreaded disease.
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Affiliation(s)
- Kuan Jiang
- Mechanobiology Institute, National University of Singapore, Singapore
| | - Lanfeng Liang
- Mechanobiology Institute, National University of Singapore, Singapore
| | - Chwee Teck Lim
- Mechanobiology Institute, National University of Singapore, Singapore
- Department of Biomedical Engineering, National University of Singapore, Singapore
- Institute for Health Innovation and Technology (iHealthtech), National University of Singapore, Singapore
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11
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Wolf KJ, Shukla P, Springer K, Lee S, Coombes JD, Choy CJ, Kenny SJ, Xu K, Kumar S. A mode of cell adhesion and migration facilitated by CD44-dependent microtentacles. Proc Natl Acad Sci U S A 2020; 117:11432-11443. [PMID: 32381732 PMCID: PMC7261014 DOI: 10.1073/pnas.1914294117] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
The structure and mechanics of many connective tissues are dictated by a collagen-rich extracellular matrix (ECM), where collagen fibers provide topological cues that direct cell migration. However, comparatively little is known about how cells navigate the hyaluronic acid (HA)-rich, nanoporous ECM of the brain, a problem with fundamental implications for development, inflammation, and tumor invasion. Here, we demonstrate that glioblastoma cells adhere to and invade HA-rich matrix using microtentacles (McTNs), which extend tens of micrometers from the cell body and are distinct from filopodia. We observe these structures in continuous culture models and primary patient-derived tumor cells, as well as in synthetic HA matrix and organotypic brain slices. High-magnification and superresolution imaging reveals McTNs are dynamic, CD44-coated tubular protrusions containing microtubules and actin filaments, which respectively drive McTN extension and retraction. Molecular mechanistic studies reveal that McTNs are stabilized by an interplay between microtubule-driven protrusion, actomyosin-driven retraction, and CD44-mediated adhesion, where adhesive and cytoskeletal components are mechanistically coupled by an IQGAP1-CLIP170 complex. McTNs represent a previously unappreciated mechanism through which cells engage nanoporous HA matrix and may represent an important molecular target in physiology and disease.
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Affiliation(s)
- Kayla J Wolf
- University of California, Berkeley-University of California San Francisco Graduate Program in Bioengineering, Department of Bioengineering, University of California, Berkeley, CA 94720
- Department of Bioengineering, University of California, Berkeley, CA, 94720
| | - Poojan Shukla
- Department of Bioengineering, University of California, Berkeley, CA, 94720
| | - Kelsey Springer
- Department of Bioengineering, University of California, Berkeley, CA, 94720
| | - Stacey Lee
- University of California, Berkeley-University of California San Francisco Graduate Program in Bioengineering, Department of Bioengineering, University of California, Berkeley, CA 94720
- Department of Bioengineering, University of California, Berkeley, CA, 94720
| | - Jason D Coombes
- Department of Bioengineering, University of California, Berkeley, CA, 94720
- Inflammation Biology, School of Immunology and Microbial Sciences, Faculty of Life Sciences and Medicine, King's College London, London, United Kingdom, SE5 9NU
| | - Caleb J Choy
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
| | - Samuel J Kenny
- Department of Chemistry, University of California, Berkeley, CA 94720
| | - Ke Xu
- Department of Chemistry, University of California, Berkeley, CA 94720
- Division of Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720
| | - Sanjay Kumar
- University of California, Berkeley-University of California San Francisco Graduate Program in Bioengineering, Department of Bioengineering, University of California, Berkeley, CA 94720;
- Department of Bioengineering, University of California, Berkeley, CA, 94720
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720
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12
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Lee K, Park TIH, Heppner P, Schweder P, Mee EW, Dragunow M, Montgomery JM. Human in vitro systems for examining synaptic function and plasticity in the brain. J Neurophysiol 2020; 123:945-965. [PMID: 31995449 DOI: 10.1152/jn.00411.2019] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The human brain shows remarkable complexity in its cellular makeup and function, which are distinct from nonhuman species, signifying the need for human-based research platforms for the study of human cellular neurophysiology and neuropathology. However, the use of adult human brain tissue for research purposes is hampered by technical, methodological, and accessibility challenges. One of the major problems is the limited number of in vitro systems that, in contrast, are readily available from rodent brain tissue. With recent advances in the optimization of protocols for adult human brain preparations, there is a significant opportunity for neuroscientists to validate their findings in human-based systems. This review addresses the methodological aspects, advantages, and disadvantages of human neuron in vitro systems, focusing on the unique properties of human neurons and synapses in neocortical microcircuits. These in vitro models provide the incomparable advantage of being a direct representation of the neurons that have formed part of the human brain until the point of recording, which cannot be replicated by animal models nor human stem-cell systems. Important distinct cellular mechanisms are observed in human neurons that may underlie the higher order cognitive abilities of the human brain. The use of human brain tissue in neuroscience research also raises important ethical, diversity, and control tissue limitations that need to be considered. Undoubtedly however, these human neuron systems provide critical information to increase the potential of translation of treatments from the laboratory to the clinic in a way animal models are failing to provide.
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Affiliation(s)
- Kevin Lee
- Department of Physiology, University of Auckland, Auckland, New Zealand.,Centre for Brain Research, University of Auckland, New Zealand
| | - Thomas I-H Park
- Centre for Brain Research, University of Auckland, New Zealand.,Department of Pharmacology, University of Auckland, Auckland, New Zealand
| | - Peter Heppner
- Centre for Brain Research, University of Auckland, New Zealand.,Department of Neurosurgery, Auckland City Hospital, Auckland, New Zealand
| | - Patrick Schweder
- Centre for Brain Research, University of Auckland, New Zealand.,Department of Neurosurgery, Auckland City Hospital, Auckland, New Zealand
| | - Edward W Mee
- Centre for Brain Research, University of Auckland, New Zealand.,Department of Neurosurgery, Auckland City Hospital, Auckland, New Zealand
| | - Michael Dragunow
- Centre for Brain Research, University of Auckland, New Zealand.,Department of Pharmacology, University of Auckland, Auckland, New Zealand
| | - Johanna M Montgomery
- Department of Physiology, University of Auckland, Auckland, New Zealand.,Centre for Brain Research, University of Auckland, New Zealand
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13
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da Hora CC, Schweiger MW, Wurdinger T, Tannous BA. Patient-Derived Glioma Models: From Patients to Dish to Animals. Cells 2019; 8:E1177. [PMID: 31574953 PMCID: PMC6829406 DOI: 10.3390/cells8101177] [Citation(s) in RCA: 82] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Revised: 09/13/2019] [Accepted: 09/27/2019] [Indexed: 02/07/2023] Open
Abstract
Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults associated with a poor survival. Current standard of care consists of surgical resection followed by radiation and chemotherapy. GBMs are highly heterogeneous, having a complex interaction among different cells within the tumor as well as the tumor microenvironment. One of the main challenges in the neuro-oncology field in general, and GBM in particular, is to find an optimum culture condition that maintains the molecular genotype and phenotype as well as heterogeneity of the original tumor in vitro and in vivo. Established cell lines were shown to be a poor model of the disease, failing to recapitulate the phenotype and harboring non-parental genotypic mutations. Given the growing understanding of GBM biology, the discovery of glioma cancer stem-like cells (GSCs), and their role in tumor formation and therapeutic resistance, scientists are turning more towards patient-derived cells and xenografts as a more representative model. In this review, we will discuss the current state of patient-derived GSCs and their xenografts; and provide an overview of different established models to study GBM biology and to identify novel therapeutics in the pre-clinical phase.
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Affiliation(s)
- Cintia Carla da Hora
- Experimental Therapeutics and Molecular Imaging Laboratory, Department of Neurology, Neuro-Oncology Division, Massachusetts General Hospital, Boston, MA 02129, USA
- Neuroscience Program, Harvard Medical School, Boston MA 02129, USA
- Department of Neurosurgery, Cancer Center Amsterdam, Brain Tumor Center Amsterdam, Amsterdam UMC, Vrije Universiteit Medical Center, 1081 HV Amsterdam, The Netherlands
| | - Markus W Schweiger
- Experimental Therapeutics and Molecular Imaging Laboratory, Department of Neurology, Neuro-Oncology Division, Massachusetts General Hospital, Boston, MA 02129, USA
- Neuroscience Program, Harvard Medical School, Boston MA 02129, USA
- Department of Neurosurgery, Cancer Center Amsterdam, Brain Tumor Center Amsterdam, Amsterdam UMC, Vrije Universiteit Medical Center, 1081 HV Amsterdam, The Netherlands
| | - Thomas Wurdinger
- Department of Neurosurgery, Cancer Center Amsterdam, Brain Tumor Center Amsterdam, Amsterdam UMC, Vrije Universiteit Medical Center, 1081 HV Amsterdam, The Netherlands
| | - Bakhos A Tannous
- Experimental Therapeutics and Molecular Imaging Laboratory, Department of Neurology, Neuro-Oncology Division, Massachusetts General Hospital, Boston, MA 02129, USA.
- Neuroscience Program, Harvard Medical School, Boston MA 02129, USA.
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14
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Ravi VM, Joseph K, Wurm J, Behringer S, Garrelfs N, d'Errico P, Naseri Y, Franco P, Meyer-Luehmann M, Sankowski R, Shah MJ, Mader I, Delev D, Follo M, Beck J, Schnell O, Hofmann UG, Heiland DH. Human organotypic brain slice culture: a novel framework for environmental research in neuro-oncology. Life Sci Alliance 2019; 2:2/4/e201900305. [PMID: 31249133 PMCID: PMC6599970 DOI: 10.26508/lsa.201900305] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2019] [Revised: 06/13/2019] [Accepted: 06/14/2019] [Indexed: 12/18/2022] Open
Abstract
When it comes to the human brain, models that closely mimic in vivo conditions are lacking. Living neuronal tissue is the closest representation of the in vivo human brain outside of a living person. Here, we present a method that can be used to maintain therapeutically resected healthy neuronal tissue for prolonged periods without any discernible changes in tissue vitality, evidenced by immunohistochemistry, genetic expression, and electrophysiology. This method was then used to assess glioblastoma (GBM) progression in its natural environment by microinjection of patient-derived tumor cells into cultured sections. The result closely resembles the pattern of de novo tumor growth and invasion, drug therapy response, and cytokine environment. Reactive transformation of astrocytes, as an example of the cellular nonmalignant tumor environment, can be accurately simulated with transcriptional differences similar to those of astrocytes isolated from acute GBM specimens. In a nutshell, we present a simple method to study GBM in its physiological environment, from which valuable insights can be gained. This technique can lead to further advancements in neuroscience, neuro-oncology, and pharmacotherapy.
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Affiliation(s)
- Vidhya M Ravi
- Translational NeuroOncology Research Group, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany .,Neuroelectronic Systems, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Kevin Joseph
- Translational NeuroOncology Research Group, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Julian Wurm
- Translational NeuroOncology Research Group, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Simon Behringer
- Translational NeuroOncology Research Group, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Nicklas Garrelfs
- Translational NeuroOncology Research Group, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Paolo d'Errico
- Department of Neurology, Medical Centre, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Yashar Naseri
- Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Pamela Franco
- Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Melanie Meyer-Luehmann
- Department of Neurology, Medical Centre, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Roman Sankowski
- Institute of Neuropathology, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Mukesch Johannes Shah
- Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Irina Mader
- Clinic for Neuropediatrics and Neurorehabilitation, Epilepsy Center for Children and Adolescents, Schön Klinik, Vogtareuth, Germany
| | - Daniel Delev
- Department of Neurosurgery, University of Aachen, Aachen, Germany
| | - Marie Follo
- Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany.,Department of Medicine I, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany
| | - Jürgen Beck
- Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Oliver Schnell
- Translational NeuroOncology Research Group, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Ulrich G Hofmann
- Neuroelectronic Systems, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
| | - Dieter Henrik Heiland
- Translational NeuroOncology Research Group, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany .,Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany.,Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany
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15
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Tamura R, Miyoshi H, Sampetrean O, Shinozaki M, Morimoto Y, Iwasawa C, Fukaya R, Mine Y, Masuda H, Maruyama T, Narita M, Saya H, Yoshida K, Okano H, Toda M. Visualization of spatiotemporal dynamics of human glioma stem cell invasion. Mol Brain 2019; 12:45. [PMID: 31060588 PMCID: PMC6503361 DOI: 10.1186/s13041-019-0462-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2019] [Accepted: 04/16/2019] [Indexed: 12/11/2022] Open
Abstract
Glioblastoma exhibits phenotypic and genetic heterogeneity, aggressive invasiveness, therapeutic resistance, and tumor recurrence, which can be explained by the existence of glioma stem cells (GSCs). In this study, we visualized the spatiotemporal dynamics of invasion of human GSCs in an orthotopic xenograft mouse model using time-lapse imaging of organotypic brain slice cultures and three-dimensional imaging of optically cleared whole brains. GSCs implanted in the striatum exhibited directional migration toward axon bundles, perivascular area, and the subventricular zone around the inferior horn of the lateral ventricle. GSCs migrated in a helical pattern around axon bundles in the striatum and invaded broadly in both the rostral and caudal directions. GSCs in the corpus callosum migrated more rapidly and unidirectionally toward the contralateral side with pseudopod extension. These characteristics of GSC invasion shared histological features observed in glioblastoma patients. Spatiotemporal visualization techniques can contribute to the elucidation of the mechanisms underlying GSC invasion that may lead to the development of effective therapy for glioblastoma.
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Affiliation(s)
- Ryota Tamura
- Department of Neurosurgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Hiroyuki Miyoshi
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.
| | - Oltea Sampetrean
- Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Munehisa Shinozaki
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Yukina Morimoto
- Department of Neurosurgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Chizuru Iwasawa
- Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo, 142-8501, Japan
| | - Raita Fukaya
- Department of Neurosurgery, Fuji Hospital, 137-1 Nishiyashiki, Chiryu-shi, Aichi, 472-0007, Japan
| | - Yutaka Mine
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Hirotaka Masuda
- Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Tetsuo Maruyama
- Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Minoru Narita
- Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo, 142-8501, Japan
| | - Hideyuki Saya
- Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Kazunari Yoshida
- Department of Neurosurgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Masahiro Toda
- Department of Neurosurgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.
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16
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Kurogi R, Nakamizo A, Suzuki SO, Mizoguchi M, Yoshimoto K, Amano T, Amemiya T, Takagishi S, Iihara K. Inhibition of glioblastoma cell invasion by hsa-miR-145-5p and hsa-miR-31-5p co-overexpression in human mesenchymal stem cells. J Neurosurg 2019; 130:44-55. [PMID: 29521593 DOI: 10.3171/2017.8.jns1788] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2017] [Accepted: 08/28/2017] [Indexed: 01/09/2023]
Abstract
OBJECTIVE Human bone marrow–derived mesenchymal stem cells (hMSCs) show tropism for brain tumors and may be a useful vehicle for drug or gene delivery to malignant gliomas. Recently, some microRNAs (miRNAs) have been shown to suppress the invasiveness of malignant gliomas. METHODS To test their potential to become vehicles for the delivery of miRNA to malignant gliomas, hMSCs were engineered so that hMSC secretion of miRNAs that inhibit glioma cell invasion was enabled without altering the hMSC tropism for glioma cells. RESULTS In coculture, hMSCs cotransfected with hsa-miR-145-5p and -31-5p miRNAs showed markedly reduced invasion by U87 glioma cells in a contact-dependent manner both in vitro and ex vivo, with invasion of hMSCs cotransfected with these 2 miRNAs by the U87 cells reduced to 60.7% compared with control cells. According to a Matrigel invasion assay, the tropism of the hMSCs for U87 cells was not affected. In glioma cell lines U251 and LN229, hMSCs exhibited tropism in vivo, and invasion of hMSCs cotransfected with hsa-miR-145-5p and -31-5p was also significantly less than that of control cells. When U87 cells were coimplanted into the striatum of organotypic rat brain slices with hMSCs cotransfected with hsa-miR-145 and -31-5p, the relative invasive area decreased by 37.1%; interestingly, these U87 cells showed a change to a rounded morphology that was apparent at the invasion front. Whole-genome microarray analysis of the expression levels of 58,341 genes revealed that the co-overexpression of hsa-miR-145-5p and -31-5p downregulated FSCN1 expression in U87 cells. CONCLUSIONS This study demonstrates that miRNA overexpression in hMSCs can alter the function of glioma cells via contact-dependent transfer. Co-overexpression of multiple miRNAs may be a useful and novel therapeutic strategy. The study results suggest that hMSCs can be applied as a delivery vehicle for miRNAs.
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Affiliation(s)
- Ryota Kurogi
- Departments of1Neurosurgery and
- 2Department of Neurosurgery, National Hospital Organization, Clinical Research Institute, Kyushu Medical Center, Fukuoka, Japan
| | - Akira Nakamizo
- Departments of1Neurosurgery and
- 2Department of Neurosurgery, National Hospital Organization, Clinical Research Institute, Kyushu Medical Center, Fukuoka, Japan
| | - Satoshi O Suzuki
- 3Neuropathology, Graduate School of Medical Sciences, Kyushu University; and
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17
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Park JY, Park DH, Jeon Y, Kim YJ, Lee J, Shin MS, Kang KS, Hwang GS, Kim HY, Yamabe N. Eupatilin inhibits angiogenesis-mediated human hepatocellular metastasis by reducing MMP-2 and VEGF signaling. Bioorg Med Chem Lett 2018; 28:3150-3154. [PMID: 30177376 DOI: 10.1016/j.bmcl.2018.08.034] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2018] [Revised: 08/23/2018] [Accepted: 08/27/2018] [Indexed: 12/18/2022]
Abstract
Metastasis is responsible for the great majority of deaths in cancer patients. Matrix metalloproteinases (MMPs) have critical functions in cancer metastasis. Especially, MMP-2 and MMP-9 play a major role in tumor-cell migration and invasion. Therefore, to first find out the inhibitory effect of eupatilin on expression of MMPs in SNU182 cells, we used quantitative real-rime PCR to measure MMP-2 and MMP-9 mRNA levels. Eupatilin suppressed transcription of MMP-2 in SNU182 cells more than did the corresponding controls. Also, eupatilin significantly blocked tube formation when treated with a concentration of 3.125 or 6.25 μg/mL on human umbilical vein vascular endothelial cells (HUVECs). Eupatilin induced significant anti-angiogenic potential associated with down-regulation of hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), and phosphorylated Akt expression. Thus, tube-formation inhibition and MMP-2-mediated migration are likely to be important therapeutic targets of eupatilin in hepatocellular carcinoma metastasis.
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Affiliation(s)
- Jun Yeon Park
- Department of Food Science and Biotechnology, Kyonggi University, Suwon 16227, Republic of Korea
| | - Do Hwi Park
- College of Korean Medicine, Gachon University, Seongnam 13120, Republic of Korea
| | - Youngsic Jeon
- Department of Pathology, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea
| | - Young-Joo Kim
- Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, Gangwon-do, Republic of Korea
| | - Jaemin Lee
- College of Korean Medicine, Gachon University, Seongnam 13120, Republic of Korea
| | - Myoung-Sook Shin
- College of Korean Medicine, Gachon University, Seongnam 13120, Republic of Korea
| | - Ki Sung Kang
- College of Korean Medicine, Gachon University, Seongnam 13120, Republic of Korea
| | - Gwi Seo Hwang
- College of Korean Medicine, Gachon University, Seongnam 13120, Republic of Korea
| | - Hyun Young Kim
- Department of Food Science, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea.
| | - Noriko Yamabe
- College of Korean Medicine, Gachon University, Seongnam 13120, Republic of Korea.
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18
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de Gooijer MC, Guillén Navarro M, Bernards R, Wurdinger T, van Tellingen O. An Experimenter's Guide to Glioblastoma Invasion Pathways. Trends Mol Med 2018; 24:763-780. [PMID: 30072121 DOI: 10.1016/j.molmed.2018.07.003] [Citation(s) in RCA: 91] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2018] [Revised: 07/03/2018] [Accepted: 07/06/2018] [Indexed: 12/25/2022]
Abstract
Glioblastoma is a highly aggressive brain tumor that is characterized by its unparalleled invasiveness. Invasive glioblastoma cells not only escape surgery and focal therapies but also are more resistant to current radio- and chemo-therapeutic approaches. Thus, any curative therapy for this deadly disease likely should include treatment strategies that interfere with glioblastoma invasiveness. Understanding glioblastoma invasion mechanisms is therefore critical. We discuss the strengths and weaknesses of various glioblastoma invasion models and conclude that robust experimental evidence has been obtained for a pro-invasive role of Ephrin receptors, Rho GTPases, and casein kinase 2 (CK2). Extensive interplay occurs between these proteins, suggesting the existence of a glioblastoma invasion signaling network that comprises several targets for therapy.
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Affiliation(s)
- Mark C de Gooijer
- Division of Pharmacology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; These authors contributed equally to this work
| | - Miriam Guillén Navarro
- Division of Pharmacology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; These authors contributed equally to this work
| | - Rene Bernards
- Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
| | - Thomas Wurdinger
- Department of Neurosurgery, VU University Medical Center, Cancer Center Amsterdam, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands
| | - Olaf van Tellingen
- Division of Pharmacology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
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19
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The mode and dynamics of glioblastoma cell invasion into a decellularized tissue-derived extracellular matrix-based three-dimensional tumor model. Sci Rep 2018; 8:4608. [PMID: 29545552 PMCID: PMC5854588 DOI: 10.1038/s41598-018-22681-3] [Citation(s) in RCA: 112] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2017] [Accepted: 02/21/2018] [Indexed: 12/13/2022] Open
Abstract
Glioblastoma multiforme (GBM) is the most common brain tumor with very aggressive and infiltrative. Extracellular matrix (ECM) plays pivotal roles in the infiltrative characteristics of GBM. To understand the invasive characteristic of GBM, it is necessary to study cell-ECM interaction in the physiologically relevant biomimetic model that recapitulates the GBM-specific ECM microenvironment. Here, we propose biomimetic GBM-specific ECM microenvironment for studying mode and dynamics of glioblastoma cell invasion. Using tissue decellularization process, we constructed a patient tissue-derived ECM (pdECM)-based three-dimensional in vitro model. In our model, GBM cells exhibited heterogeneous morphology and altered the invasion routes in a microenvironment-adaptive manner. We further elucidate the effects of inhibition of ECM remodeling-related enzymatic activity (Matrix metalloproteinase (MMP) 2/9, hyaluronan synthase (HAS)) on GBM cell invasion. Interestingly, after blocking both enzyme activity, GBM cells underwent morphological transition and switch the invasion mode. Such adaptability could render cell invasion resistant to anti-cancer target therapy. There results provide insight of how organ-specific matrix differentially regulates cancer cell phenotype, and have significant implications for the design of matrix with appropriate physiologically relevant properties for in vitro tumor model.
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20
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da Silva B, Mathew RK, Polson ES, Williams J, Wurdak H. Spontaneous Glioblastoma Spheroid Infiltration of Early-Stage Cerebral Organoids Models Brain Tumor Invasion. SLAS DISCOVERY 2018. [PMID: 29543559 DOI: 10.1177/2472555218764623] [Citation(s) in RCA: 63] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Organoid methodology provides a platform for the ex vivo investigation of the cellular and molecular mechanisms underlying brain development and disease. The high-grade brain tumor glioblastoma multiforme (GBM) is considered a cancer of unmet clinical need, in part due to GBM cell infiltration into healthy brain parenchyma, making complete surgical resection improbable. Modeling the process of GBM invasion in real time is challenging as it requires both tumor and neural tissue compartments. Here, we demonstrate that human GBM spheroids possess the ability to spontaneously infiltrate early-stage cerebral organoids (eCOs). The resulting formation of hybrid organoids demonstrated an invasive tumor phenotype that was distinct from noncancerous adult neural progenitor (NP) spheroid incorporation into eCOs. These findings provide a basis for the modeling and quantification of the GBM infiltration process using a stem-cell-based organoid approach, and may be used for the identification of anti-GBM invasion strategies.
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Affiliation(s)
- Bárbara da Silva
- 1 Stem Cells and Brain Tumour Group, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, UK
| | - Ryan K Mathew
- 1 Stem Cells and Brain Tumour Group, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, UK.,2 Department of Neurosurgery, Leeds General Infirmary, Leeds, UK
| | - Euan S Polson
- 1 Stem Cells and Brain Tumour Group, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, UK
| | - Jennifer Williams
- 1 Stem Cells and Brain Tumour Group, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, UK
| | - Heiko Wurdak
- 1 Stem Cells and Brain Tumour Group, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, UK
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21
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Eisemann T, Costa B, Strelau J, Mittelbronn M, Angel P, Peterziel H. An advanced glioma cell invasion assay based on organotypic brain slice cultures. BMC Cancer 2018; 18:103. [PMID: 29378533 PMCID: PMC5789681 DOI: 10.1186/s12885-018-4007-4] [Citation(s) in RCA: 65] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2017] [Accepted: 01/18/2018] [Indexed: 01/08/2023] Open
Abstract
BACKGROUND The poor prognosis for glioblastoma patients is caused by the diffuse infiltrative growth pattern of the tumor. Therefore, the molecular and cellular processes underlying cell migration continue to be a major focus of glioblastoma research. Emerging evidence supports the concept that the tumor microenvironment has a profound influence on the functional properties of tumor cells. Accordingly, substantial effort must be devoted to move from traditional two-dimensional migration assays to three-dimensional systems that more faithfully recapitulate the complex in vivo tumor microenvironment. METHODS In order to mimic the tumor microenvironment of adult gliomas, we used adult organotypic brain slices as an invasion matrix for implanted, fluorescently labeled tumor spheroids. Cell invasion was imaged by confocal or epi-fluorescence microscopy and quantified by determining the average cumulative sprout length per spheroid. The tumor microenvironment was manipulated by treatment of the slice with small molecule inhibitors or using different genetically engineered mouse models as donors. RESULTS Both epi-fluorescence and confocal microscopy were applied to precisely quantify cell invasion in this ex vivo approach. Usage of a red-emitting membrane dye in addition to tissue clearing drastically improved epi-fluorescence imaging. Preparation of brain slices from of a genetically engineered mouse with a loss of a specific cell surface protein resulted in significantly impaired tumor cell invasion. Furthermore, jasplakinolide treatment of either tumor cells or brain slice significantly reduced tumor cell invasion. CONCLUSION We present an optimized invasion assay that closely reflects in vivo invasion by the implantation of glioma cells into organotypic adult brain slice cultures with a preserved cytoarchitecture. The diversity of applications including manipulation of the tumor cells as well as the microenvironment, permits the investigation of rate limiting factors of cell migration in a reliable context. This model will be a valuable tool for the discovery of the molecular mechanisms underlying glioma cell invasion and, ultimately, the development of novel therapeutic strategies.
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Affiliation(s)
- Tanja Eisemann
- Division of Signal Transduction and Growth Control, DKFZ/ZMBH Alliance, Heidelberg, Germany
| | - Barbara Costa
- Division of Signal Transduction and Growth Control, DKFZ/ZMBH Alliance, Heidelberg, Germany
| | - Jens Strelau
- Functional Neuroanatomy, University of Heidelberg, Heidelberg, Germany
| | - Michel Mittelbronn
- Institute of Neurology (Edinger-Institute), University Hospital Frankfurt, Goethe University, Frankfurt, Germany.,Luxembourg Centre of Neuropathology (LCNP), Dudelange, Luxembourg.,Laboratoire National de Santé, Dudelange, Luxembourg.,Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg.,Department of Oncology, NORLUX Neuro-Oncology Laboratory, Luxembourg Institute of Health (L.I.H.), Strassen, Luxembourg
| | - Peter Angel
- Division of Signal Transduction and Growth Control, DKFZ/ZMBH Alliance, Heidelberg, Germany.
| | - Heike Peterziel
- Division of Signal Transduction and Growth Control, DKFZ/ZMBH Alliance, Heidelberg, Germany.,Present address: Translational Program, Hopp Children's Cancer Center at NCT Heidelberg (KiTZ), University Hospital and DKFZ Heidelberg, Heidelberg, Germany.,Present address: Clinical Cooperation Unit Pediatric Oncology, DKFZ, Heidelberg, Germany.,German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany
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22
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Role of Microenvironment in Glioma Invasion: What We Learned from In Vitro Models. Int J Mol Sci 2018; 19:ijms19010147. [PMID: 29300332 PMCID: PMC5796096 DOI: 10.3390/ijms19010147] [Citation(s) in RCA: 82] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2017] [Revised: 12/30/2017] [Accepted: 12/31/2017] [Indexed: 12/21/2022] Open
Abstract
The invasion properties of glioblastoma hamper a radical surgery and are responsible for its recurrence. Understanding the invasion mechanisms is thus critical to devise new therapeutic strategies. Therefore, the creation of in vitro models that enable these mechanisms to be studied represents a crucial step. Since in vitro models represent an over-simplification of the in vivo system, in these years it has been attempted to increase the level of complexity of in vitro assays to create models that could better mimic the behaviour of the cells in vivo. These levels of complexity involved: 1. The dimension of the system, moving from two-dimensional to three-dimensional models; 2. The use of microfluidic systems; 3. The use of mixed cultures of tumour cells and cells of the tumour micro-environment in order to mimic the complex cross-talk between tumour cells and their micro-environment; 4. And the source of cells used in an attempt to move from commercial lines to patient-based models. In this review, we will summarize the evidence obtained exploring these different levels of complexity and highlighting advantages and limitations of each system used.
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Onken J, Torka R, Korsing S, Radke J, Krementeskaia I, Nieminen M, Bai X, Ullrich A, Heppner F, Vajkoczy P. Inhibiting receptor tyrosine kinase AXL with small molecule inhibitor BMS-777607 reduces glioblastoma growth, migration, and invasion in vitro and in vivo. Oncotarget 2017; 7:9876-89. [PMID: 26848524 PMCID: PMC4891090 DOI: 10.18632/oncotarget.7130] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2015] [Accepted: 01/19/2016] [Indexed: 01/28/2023] Open
Abstract
Purpose Receptor tyrosine kinase AXL (RTK-AXL) is regarded as suitable target in glioma therapy. Here we evaluate the anti-tumoral effect of small molecule inhibitor BMS-777607 targeting RTK-AXL in a preclinical glioma model and provide evidence that RTK-AXL is expressed and phosphorylated in primary and recurrent glioblastoma multiforme (GBM). Experimental design We studied the impact of BMS-777607 targeting RTK-AXL in GBM models in vitro and in vivo utilizing glioma cells SF126 and U118MG. Impact on proliferation, apoptosis and angiogenesis was investigated by immunohistochemistry (IHC) and functional assays in vitro and in vivo. Tumor growth was assessed with MRI. Human GBM tissue was analyzed in terms of RTK-AXL phosphorylation by immunoprecipitation and immunohistochemistry. Results BMS-777607 displayed various anti-cancer effects dependent on increased apoptosis, decreased proliferation and migration in vitro and ex vivo in SF126 and U118 GBM cells. In vivo we observed a 56% tumor volume reduction in SF126 xenografts and remission in U118MG xenografts of more than 91%. The tube formation assay confirmed the anti-angiogenic effect of BMS-777607, which became also apparent in tumor xenografts. IHC of human GBM tissue localized phosphorylated RTK-AXL in hypercellular tumor regions, the migratory front of tumor cells in pseudo-palisades, and in vascular proliferates within the tumor. We further proved RTK-AXL phosphorylation in primary and recurrent disease state. Conclusion Collectively, these data strongly suggest that targeting RTK-AXL with BMS-777607 could represent a novel and potent regimen for the treatment of primary and recurrent GBM.
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Affiliation(s)
- Julia Onken
- Department of Neurosurgery, Charité, Berlin, Germany
| | - Robert Torka
- Department of Molecular Biology, Max-Planck Institute of Biochemistry, Martinsried, Germany
| | - Sören Korsing
- Department of Neurosurgery, Charité, Berlin, Germany
| | | | | | | | - Xi Bai
- Department of Neurosurgery, Charité, Berlin, Germany
| | - Axel Ullrich
- Department of Molecular Biology, Max-Planck Institute of Biochemistry, Martinsried, Germany
| | - Frank Heppner
- Institute of Neuropathology, Charité, Berlin, Germany
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24
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Ivanov DP, Coyle B, Walker DA, Grabowska AM. In vitro models of medulloblastoma: Choosing the right tool for the job. J Biotechnol 2016; 236:10-25. [PMID: 27498314 DOI: 10.1016/j.jbiotec.2016.07.028] [Citation(s) in RCA: 166] [Impact Index Per Article: 18.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2016] [Accepted: 07/29/2016] [Indexed: 02/06/2023]
Abstract
The recently-defined four molecular subgroups of medulloblastoma have required updating of our understanding of in vitro models to include molecular classification and risk stratification features from clinical practice. This review seeks to build a more comprehensive picture of the in vitro systems available for modelling medulloblastoma. The subtype classification and molecular characterisation for over 40 medulloblastoma cell-lines has been compiled, making it possible to identify the strengths and weaknesses in current model systems. Less than half (18/44) of established medulloblastoma cell-lines have been subgrouped. The majority of the subgrouped cell-lines (11/18) are Group 3 with MYC-amplification. SHH cell-lines are the next most common (4/18), half of which exhibit TP53 mutation. WNT and Group 4 subgroups, accounting for 50% of patients, remain underrepresented with 1 and 2 cell-lines respectively. In vitro modelling relies not only on incorporating appropriate tumour cells, but also on using systems with the relevant tissue architecture and phenotype as well as normal tissues. Novel ways of improving the clinical relevance of in vitro models are reviewed, focusing on 3D cell culture, extracellular matrix, co-cultures with normal cells and organotypic slices. This paper champions the establishment of a collaborative online-database and linked cell-bank to catalyse preclinical medulloblastoma research.
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Affiliation(s)
- Delyan P Ivanov
- Division of Cancer and Stem Cells, Cancer Biology, University of Nottingham, Nottingham, UK.
| | - Beth Coyle
- Children's Brain Tumour Research Centre, Queens Medical Centre, University of Nottingham, Nottingham, UK.
| | - David A Walker
- Children's Brain Tumour Research Centre, Queens Medical Centre, University of Nottingham, Nottingham, UK.
| | - Anna M Grabowska
- Division of Cancer and Stem Cells, Cancer Biology, University of Nottingham, Nottingham, UK.
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25
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Li S, Li C, Ryu HH, Lim SH, Jang WY, Jung S. Bacitracin Inhibits the Migration of U87-MG Glioma Cells via Interferences of the Integrin Outside-in Signaling Pathway. J Korean Neurosurg Soc 2016; 59:106-16. [PMID: 26962415 PMCID: PMC4783475 DOI: 10.3340/jkns.2016.59.2.106] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2015] [Revised: 01/15/2016] [Accepted: 01/21/2016] [Indexed: 11/27/2022] Open
Abstract
Objective Protein disulfide isomerase (PDI) acts as a chaperone on the cell surface, and it has been reported that PDI is associated with the tumor cell migration and invasion. The aims of this study are to investigate the anti-migration effect of bacitracin, which is an inhibitor of PDI, and the associated factor in this process. Methods U87-MG glioma cells were treated with bacitracin in 1.25, 2.5, 3.75, and 5.0 mM concentrations. Western blot with caspase-3 was applied to evaluate the cytotoxicity of bacitracin. Adhesion, morphology, migration assays, and organotypic brain-slice culture were performed to evaluate the effect of bacitracin to the tumor cell. Western blot, PCR, and gelatin zymography were performed to investigate the associated factors. Thirty glioma tissues were collected following immunohistochemistry and Western blot. Results Bacitracin showed a cytotoxicity in 3rd (p<0.05) and 4th (p<0.001) days, in 5.0 Mm concentration. The cell adhesion significantly decreased and the cells became a round shape after treated with bacitracin. The migration ability, the expression of phosphorylated focal adhesion kinase (p-FAK) and matrix metalloproteinase-2 (MMP-2) decreased in a bacitracin dose- and time-dependent manner. The U87-MG cells exhibited low-invasiveness in the 2.5 mM, compared with the untreated in organotypic brain-slice culture. PDI was expressed in the tumor margin, and significantly increased with histological glioma grades (p<0.001). Conclusion Bacitracin, as a functional inhibitor of PDI, decreased the phosphorylated FAK and the secreted MMP-2, which are the downstream of integrin and play a major role in cell migration and invasion, might become one of the feasible therapeutic strategies for glioblastoma.
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Affiliation(s)
- Songyuan Li
- Brain Tumor Research Laboratory and Department of Neurosurgery Chonnam National University Research Institute of Medical Sciences, Chonnam National University Hwasun Hopital and Medical School, Hwasun, Korea
| | - Chunhao Li
- Brain Tumor Research Laboratory and Department of Neurosurgery Chonnam National University Research Institute of Medical Sciences, Chonnam National University Hwasun Hopital and Medical School, Hwasun, Korea
| | - Hyang-Hwa Ryu
- Brain Tumor Research Laboratory and Department of Neurosurgery Chonnam National University Research Institute of Medical Sciences, Chonnam National University Hwasun Hopital and Medical School, Hwasun, Korea
| | - Sa-Hoe Lim
- Brain Tumor Research Laboratory and Department of Neurosurgery Chonnam National University Research Institute of Medical Sciences, Chonnam National University Hwasun Hopital and Medical School, Hwasun, Korea
| | - Woo-Youl Jang
- Brain Tumor Research Laboratory and Department of Neurosurgery Chonnam National University Research Institute of Medical Sciences, Chonnam National University Hwasun Hopital and Medical School, Hwasun, Korea
| | - Shin Jung
- Brain Tumor Research Laboratory and Department of Neurosurgery Chonnam National University Research Institute of Medical Sciences, Chonnam National University Hwasun Hopital and Medical School, Hwasun, Korea
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van Marion DM, Domanska UM, Timmer-Bosscha H, Walenkamp AM. Studying cancer metastasis: Existing models, challenges and future perspectives. Crit Rev Oncol Hematol 2016; 97:107-17. [DOI: 10.1016/j.critrevonc.2015.08.009] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2015] [Accepted: 08/05/2015] [Indexed: 02/03/2023] Open
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Human brain slices for epilepsy research: Pitfalls, solutions and future challenges. J Neurosci Methods 2015; 260:221-32. [PMID: 26434706 DOI: 10.1016/j.jneumeth.2015.09.021] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2015] [Revised: 09/21/2015] [Accepted: 09/23/2015] [Indexed: 12/17/2022]
Abstract
Increasingly, neuroscientists are taking the opportunity to use live human tissue obtained from elective neurosurgical procedures for electrophysiological studies in vitro. Access to this valuable resource permits unique studies into the network dynamics that contribute to the generation of pathological electrical activity in the human epileptic brain. Whilst this approach has provided insights into the mechanistic features of electrophysiological patterns associated with human epilepsy, it is not without technical and methodological challenges. This review outlines the main difficulties associated with working with epileptic human brain slices from the point of collection, through the stages of preparation, storage and recording. Moreover, it outlines the limitations, in terms of the nature of epileptic activity that can be observed in such tissue, in particular, the rarity of spontaneous ictal discharges, we discuss manipulations that can be utilised to induce such activity. In addition to discussing conventional electrophysiological techniques that are routinely employed in epileptic human brain slices, we review how imaging and multielectrode array recordings could provide novel insights into the network dynamics of human epileptogenesis. Acute studies in human brain slices are ultimately limited by the lifetime of the tissue so overcoming this issue provides increased opportunity for information gain. We review the literature with respect to organotypic culture techniques that may hold the key to prolonging the viability of this material. A combination of long-term culture techniques, viral transduction approaches and electrophysiology in human brain slices promotes the possibility of large scale monitoring and manipulation of neuronal activity in epileptic microcircuits.
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Arden JD, Lavik KI, Rubinic KA, Chiaia N, Khuder SA, Howard MJ, Nestor-Kalinoski AL, Alberts AS, Eisenmann KM. Small-molecule agonists of mammalian Diaphanous-related (mDia) formins reveal an effective glioblastoma anti-invasion strategy. Mol Biol Cell 2015; 26:3704-18. [PMID: 26354425 PMCID: PMC4626057 DOI: 10.1091/mbc.e14-11-1502] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2014] [Accepted: 09/04/2015] [Indexed: 12/26/2022] Open
Abstract
Formin agonists impede the most dangerous aspect of glioblastoma—tumor spread into surrounding healthy tissue. Formin activation impairs a novel aspect of the transformed cell and informs the development of antitumor strategies for a cancer needing a more effective therapy. The extensive invasive capacity of glioblastoma (GBM) makes it resistant to surgery, radiotherapy, and chemotherapy and thus makes it lethal. In vivo, GBM invasion is mediated by Rho GTPases through unidentified downstream effectors. Mammalian Diaphanous (mDia) family formins are Rho-directed effectors that regulate the F-actin cytoskeleton to support tumor cell motility. Historically, anti-invasion strategies focused upon mDia inhibition, whereas activation remained unexplored. The recent development of small molecules directly inhibiting or activating mDia-driven F-actin assembly that supports motility allows for exploration of their role in GBM. We used the formin inhibitor SMIFH2 and mDia agonists IMM-01/-02 and mDia2-DAD peptides, which disrupt autoinhibition, to examine the roles of mDia inactivation versus activation in GBM cell migration and invasion in vitro and in an ex vivo brain slice invasion model. Inhibiting mDia suppressed directional migration and spheroid invasion while preserving intrinsic random migration. mDia agonism abrogated both random intrinsic and directional migration and halted U87 spheroid invasion in ex vivo brain slices. Thus mDia agonism is a superior GBM anti-invasion strategy. We conclude that formin agonism impedes the most dangerous GBM component—tumor spread into surrounding healthy tissue. Formin activation impairs novel aspects of transformed cells and informs the development of anti-GBM invasion strategies.
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Affiliation(s)
- Jessica D Arden
- Department of Biochemistry and Cancer Biology, University of Toledo Health Science Campus, Toledo, OH 43614
| | - Kari I Lavik
- Department of Biochemistry and Cancer Biology, University of Toledo Health Science Campus, Toledo, OH 43614
| | - Kaitlin A Rubinic
- Department of Biochemistry and Cancer Biology, University of Toledo Health Science Campus, Toledo, OH 43614
| | - Nicolas Chiaia
- Department of Neurosciences, University of Toledo Health Science Campus, Toledo, OH 43614
| | - Sadik A Khuder
- Departments of Medicine and Public Health and Homeland Security, University of Toledo Health Science Campus, Toledo, OH 43614
| | - Marthe J Howard
- Department of Neurosciences, University of Toledo Health Science Campus, Toledo, OH 43614
| | | | - Arthur S Alberts
- Laboratory of Cell Structure and Signal Integration, Van Andel Research Institute, Grand Rapids, MI 49503
| | - Kathryn M Eisenmann
- Department of Biochemistry and Cancer Biology, University of Toledo Health Science Campus, Toledo, OH 43614 )
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Khan IS, Ehtesham M. Laboratory models for central nervous system tumor stem cell research. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2015; 853:69-83. [PMID: 25895708 DOI: 10.1007/978-3-319-16537-0_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/22/2023]
Abstract
Central nervous system (CNS) tumors are complex organ systems comprising of a neoplastic component with associated vasculature, inflammatory cells, and reactive cellular and extracellular components. Research has identified a subset of cells in CNS tumors that portray defining properties of neural stem cells, namely, that of self-renewal and multi-potency. Growing evidence suggests that these tumor stem cells (TSC) play an important role in the maintenance and growth of the tumor. Furthermore, these cells have also been shown to be refractory to conventional therapy and may be crucial for tumor recurrence and metastasis. Current investigations are focusing on isolating these TSC from CNS tumors to investigate their unique biological processes. This understanding will help identify and develop more effective and comprehensive treatment strategies. This chapter provides an overview of some of the most commonly used laboratory models for CNSTSC research.
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Affiliation(s)
- Imad Saeed Khan
- Section of Neurosurgery, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA
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Zhao HW, Li YW, Feng R, Yu JB, Li J, Zhang Y, Li JC, Wang YX. TGF-β/Smad2/3 signal pathway involves in U251 cell proliferation and apoptosis. Gene 2015; 562:76-82. [PMID: 25701598 DOI: 10.1016/j.gene.2015.02.049] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2014] [Revised: 02/12/2015] [Accepted: 02/16/2015] [Indexed: 10/24/2022]
Abstract
TGF-β/Smad2/3 signal pathway is regarded as a central regulator in various tumors, but its roles in brain cancer therapy remain unknown. In this study, we identify that the TGF-β/Smad2/3 signal pathway is activated in human brain glioma cells; inhibitor (SB203580) and siRNA against Smad2/3 quickly inhibited the phosphorylation of Smad2 and 3, expression of its major downstream gene, Ki-67, arrested cells in the G2/M phase and induced apoptosis of cells. The findings suggest that TGF-β/Smad2/3 pathway plays a key role in U251 cell growth and metastasis, which suggests its potential role in the molecular therapy of brain cancer.
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Affiliation(s)
- Hong-wei Zhao
- Department of Anesthesiology, Tianjin Medical University Cancer Institute and Hospital, Key Laboratory of Cancer Prevention and Treatment of Tianjin City, Tianjin 300060, China
| | - Yan-Wei Li
- Department of Nephrology, The Second Hospital of Tianjin Medical University, Tianjin 300211, China
| | - Ren Feng
- Tianjin Huanhu Hospital, Tianjin 300060, China
| | - Jian-Bo Yu
- Department of Anesthesiology, Tianjin Nan Kai Hospital, Tianjin 300100, China
| | - Jing Li
- Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin 300052, China
| | - Yan Zhang
- Department of Anesthesiology, Tianjin Huanhu Hospital, Tianjin 300060, China.
| | - Jin-Cheng Li
- Department of Anesthesiology, Tianjin Medical University Cancer Institute and Hospital, Key Laboratory of Cancer Prevention and Treatment of Tianjin City, Tianjin 300060, China
| | - Ya-Xin Wang
- Department of Anesthesiology, Tianjin Huanhu Hospital, Tianjin 300060, China
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Eugène E, Cluzeaud F, Cifuentes-Diaz C, Fricker D, Le Duigou C, Clemenceau S, Baulac M, Poncer JC, Miles R. An organotypic brain slice preparation from adult patients with temporal lobe epilepsy. J Neurosci Methods 2014; 235:234-44. [PMID: 25064188 PMCID: PMC4426207 DOI: 10.1016/j.jneumeth.2014.07.009] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2014] [Revised: 07/13/2014] [Accepted: 07/15/2014] [Indexed: 02/05/2023]
Abstract
BACKGROUND A long-term in vitro preparation of diseased brain tissue would facilitate work on human pathologies. Organotypic tissue cultures retain an appropriate neuronal form, spatial arrangement, connectivity and electrical activity over several weeks. However, they are typically prepared with tissue from immature animals. In work using tissue from adult animals or humans, survival times longer than a few days have not been reported and it is not clear that pathological neuronal activities are retained. NEW METHOD We modified tissue preparation procedures and used a defined culture medium to make organotypic cultures of temporal lobe tissue obtained after operations on adult patients with pharmaco-resistant mesial temporal lobe epilepsies. RESULTS Organototypic culture preparation and maintenance techniques were judged on criteria of morphology and the generation of epileptiform activities. Short-duration (30-100 ms) interictal-like population activities were initiated spontaneously in either the subiculum, dentate gyrus or the CA2/CA3 region, but not the cortex, for up to 3-4 weeks in culture. Ictal-like discharges, of duration greater than 10s, were induced by convulsants. Epileptiform activities were modulated by both glutamatergic and GABAergic receptor antagonists. COMPARISON WITH EXISTING METHODS Our methods now permit the maintenance in organotypic culture of epileptic adult human tissue, generating appropriate epileptiform activity over 3-4 weeks. CONCLUSIONS We have shown that characteristic morphology and pathological activities are maintained in organotypic cultures of adult human tissue. These cultures should permit studies on the effects of prolonged drug treatments and long-term procedures such as viral transduction.
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Affiliation(s)
- Emmanuel Eugène
- Inserm U1127, CNRS UMR7225, Sorbonne Universités, UPMC Univ Paris6 UMR S1127, Institut du Cerveau et de la Moelle épinière, 47 Boulevard de l'Hôpital, Paris 75013, France; INSERM, UMR-839, Paris 75005, France; UPMC Univ Paris, UMR-839, Paris 75005, France; Institut du Fer a Moulin, Paris 75005 France.
| | - Françoise Cluzeaud
- Service Microscopie, Centre de recherche biomedicale, CHU Bichat, Université Paris Diderot, 16 rue Henri Huchard, Paris 75870, France
| | - Carmen Cifuentes-Diaz
- INSERM, UMR-839, Paris 75005, France; UPMC Univ Paris, UMR-839, Paris 75005, France; Institut du Fer a Moulin, Paris 75005 France
| | - Desdemona Fricker
- Inserm U1127, CNRS UMR7225, Sorbonne Universités, UPMC Univ Paris6 UMR S1127, Institut du Cerveau et de la Moelle épinière, 47 Boulevard de l'Hôpital, Paris 75013, France
| | - Caroline Le Duigou
- Inserm U1127, CNRS UMR7225, Sorbonne Universités, UPMC Univ Paris6 UMR S1127, Institut du Cerveau et de la Moelle épinière, 47 Boulevard de l'Hôpital, Paris 75013, France
| | - Stephane Clemenceau
- Inserm U1127, CNRS UMR7225, Sorbonne Universités, UPMC Univ Paris6 UMR S1127, Institut du Cerveau et de la Moelle épinière, 47 Boulevard de l'Hôpital, Paris 75013, France
| | - Michel Baulac
- Inserm U1127, CNRS UMR7225, Sorbonne Universités, UPMC Univ Paris6 UMR S1127, Institut du Cerveau et de la Moelle épinière, 47 Boulevard de l'Hôpital, Paris 75013, France
| | - Jean-Christophe Poncer
- INSERM, UMR-839, Paris 75005, France; UPMC Univ Paris, UMR-839, Paris 75005, France; Institut du Fer a Moulin, Paris 75005 France
| | - Richard Miles
- Inserm U1127, CNRS UMR7225, Sorbonne Universités, UPMC Univ Paris6 UMR S1127, Institut du Cerveau et de la Moelle épinière, 47 Boulevard de l'Hôpital, Paris 75013, France.
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Daviaud N, Garbayo E, Schiller PC, Perez-Pinzon M, Montero-Menei CN. Organotypic cultures as tools for optimizing central nervous system cell therapies. Exp Neurol 2013; 248:429-40. [DOI: 10.1016/j.expneurol.2013.07.012] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2013] [Revised: 07/15/2013] [Accepted: 07/18/2013] [Indexed: 01/01/2023]
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Dual effect of serum amyloid A on the invasiveness of glioma cells. Mediators Inflamm 2013; 2013:509089. [PMID: 23533307 PMCID: PMC3596950 DOI: 10.1155/2013/509089] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2012] [Revised: 01/04/2013] [Accepted: 01/22/2013] [Indexed: 02/02/2023] Open
Abstract
Evidence sustains a role for the acute-phase protein serum amyloid A (SAA) in carcinogenesis and metastasis, and the protein has been suggested as a marker for tumor progression. Nevertheless, the demonstration of a direct activity of SAA on tumor cells is still incipient. We have investigated the effect of human recombinant SAA (rSAA) on two human glioma cell lines, A172 and T98G. rSAA stimulated the [(3)H]-thymidine incorporation of both lines, but had dual effects on migration and invasiveness which varied according to the cell line. In T98G, the rSAA increased migration and invasion behaviors whereas in A172 it decreased these behaviors. These findings agree with the effect triggered by rSAA on matrix metalloproteinases (MMPs) activities measured in a gelatinolytic assay. rSAA inhibited activity of both MMPs in A172 cells while increasing them in T98G cells. rSAA also affected the production of compounds present in the tumor microenvironment that orchestrate tumor progression, such as IL-8, the production of reactive oxygen species (ROS) and nitric oxide (NO). We also observed that both lines expressed all three of the isoforms of SAA: SAA1, SAA2, and SAA4. These data suggest that some tumor cells are responsive to SAA and, in these cases, SAA may have a role in cancer progression that varies according to the cell type.
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Zimmermann M, Box C, Eccles SA. Two-dimensional vs. three-dimensional in vitro tumor migration and invasion assays. Methods Mol Biol 2013; 986:227-52. [PMID: 23436416 DOI: 10.1007/978-1-62703-311-4_15] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/12/2023]
Abstract
Motility and invasion are key hallmarks that distinguish benign from malignant tumors, enabling cells to cross tissue boundaries, disseminate in blood and lymph and establish metastases at distant sites. Similar properties are also utilized by activated endothelial cells during tumor-induced angiogenesis. It is now appreciated that these processes might provide a rich source of novel molecular targets with the potential for inhibitors to restrain both metastasis and neoangiogenesis. Such therapeutic strategies require assays that can rapidly and quantitatively measure cell movement and the ability to traverse physiological barriers. The need for high-throughput, however, must be balanced by assay designs that accommodate, as far as possible, the complexity of the in vivo tumor microenvironment. This chapter aims to give an overview of some commonly used migration and invasion assays to aid in the selection of a balanced portfolio of techniques for the rapid and accurate evaluation of novel therapeutic agents.
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Affiliation(s)
- Miriam Zimmermann
- Tumour Biology and Metastasis, Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, McElwain Laboratories, The Institute of Cancer Research, Surrey, UK
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Neurotrophin-3 modulates breast cancer cells and the microenvironment to promote the growth of breast cancer brain metastasis. Oncogene 2012; 32:4064-77. [PMID: 23001042 DOI: 10.1038/onc.2012.417] [Citation(s) in RCA: 82] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2012] [Revised: 07/16/2012] [Accepted: 07/31/2012] [Indexed: 01/17/2023]
Abstract
Metastasis, which remains incompletely characterized at the molecular and biochemical levels, is a highly specific process. Despite the ability of disseminated cancer cells to intravasate into distant tissues, it has been long recognized that only a limited subset of target organs develop clinically overt metastases. Therefore, subsequent adaptation of disseminated cancer cells to foreign tissue microenvironment determines the metastatic latency and tissue tropism of these cells. As a result, studying interactions between the disseminated cancer cells and the adjacent stromal cells will provide a better understanding of what constitutes a favorable or unfavorable microenvironment for disseminated cancer cells in a tissue-specific manner. Previously, we reported a protein signature of brain metastasis showing increased ability of brain metastatic breast cancer cells to counteract oxidative stress. In this study, we showed that another protein from the brain metastatic protein signature, neurotrophin-3 (NT-3), has a dual function of regulating the metastatic growth of metastatic breast cancer cells and reducing the activation of immune response in the brain. More importantly, increased NT-3 secretion in metastatic breast cancer cells results in a reversion of mesenchymal-like (EMT) state to epithelial-like (MET) state and vice versa. Ectopic expression of NT-3 in EMT-like breast cancer cells reduces their migratory ability and increases the expression of HER2 (human epidermal growth factor receptor 2) and E-cadherin at the cell-cell junction. In addition, both endogenous and ectopic expression of NT-3 reduced the number of fully activated cytotoxic microglia. In summary, NT-3 appears to promote growth of metastatic breast cancer cells in the brain by facilitating the re-epithelialization of metastatic breast cancer cells and downmodulating the cytotoxic response of microglia. Most importantly, our results provide new insights into the latency and development of central nervous system macrometastases in patients with HER2-positive breast tumors and provide mechanistic rationale to target HER2 signaling for HER2-positive breast cancer brain metastasis.
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Magnetic resonance imaging of solitary brain metastases: main findings of nonmorphological sequences. Radiol Med 2012; 117:1225-41. [PMID: 22744350 DOI: 10.1007/s11547-012-0846-2] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2011] [Accepted: 07/14/2011] [Indexed: 10/28/2022]
Abstract
PURPOSE This study was done to investigate the usefulness of diffusion-weighted (DWI), perfusion-weighted (PWI) and proton magnetic resonance (MR) spectroscopy imaging in characterising solitary brain metastases. MATERIALS AND METHODS Fifty-nine solitary brain metastases were evaluated with conventional and nonmorphological MR imaging: DWI, PWI and MR spectroscopy. We evaluated size, signal intensity and contrast enhancement and calculated apparent diffusion coefficient (ADC), relative cerebral blood volume (rCBV), percentage of signal intensity recovery (PSR) and maximum values of N-acetylaspartate (NAA), choline (Cho), creatine (Cr), lipids (Lip), NAA/Cr and Cho/Cr. The nonmorphological parameters were compared with those from the literature for brain lesions that frequently enter the differential diagnosis with metastases. RESULTS Signal intensity and contrast enhancement patterns were variable. There was a wide range of ADC values: min:max 0.59×10(-3):1.88×10(-3). Compared with normal white matter, rCBV was higher in lesions (3.30±1.59) and lower in perilesional oedema (0.42±0.15). Mean and minimum PSR were 57% and 48%, respectively; lip and Cho were elevated and NAA reduced. CONCLUSIONS Conventional MR findings of solitary metastases are heterogeneous, and some values of nonmorphological sequences are similar to those of other brain lesions. PWI seems to be the nonmorphological MR technique that may best contribute to the diagnosis of brain metastases.
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Shimizu F, Hovinga KE, Metzner M, Soulet D, Tabar V. Organotypic explant culture of glioblastoma multiforme and subsequent single-cell suspension. ACTA ACUST UNITED AC 2012; Chapter 3:Unit3.5. [PMID: 22135084 DOI: 10.1002/9780470151808.sc0305s19] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Glioblastoma multiforme (GBM) is one of the most aggressive brain tumors. GBM cell lines used in laboratory studies are frequently passaged in various culture media at high proliferation rates, resulting in significant genetic and molecular alterations. Thus, data obtained in cell lines are often inapplicable to patient tumors. Furthermore, recent studies suggest that there is a stem cell-like hierarchy among GBM cell populations and a crucial role for tumor vasculature in stem cells, as well as tumor growth, which cannot be reproduced in cell line cultures. Our laboratory has developed a novel three-dimensional (3D) organotypic "explant" system of surgical GBM specimens that preserves tumor cells in their original milieu, as well as the cytoarchitecture of the tumor stroma. Our previous study on the role of Notch inhibition has demonstrated a definitive effect on the tumor endothelium that could only be highlighted by this system. In this unit, we describe a detailed protocol for preparing GBM explants, and discuss strengths, as well as limitations of the explant system as an in vitro 3D model of GBM.
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Affiliation(s)
- Fumiko Shimizu
- Department of Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
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Nørregaard A, Jensen SS, Kolenda J, Aaberg-Jessen C, Christensen KG, Jensen PH, Schrøder HD, Kristensen BW. Effects of Chemotherapeutics on Organotypic Corticostriatal Slice Cultures Identified by A Panel of Fluorescent and Immunohistochemical Markers. Neurotox Res 2011; 22:43-58. [DOI: 10.1007/s12640-011-9300-9] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2011] [Revised: 12/05/2011] [Accepted: 12/06/2011] [Indexed: 11/29/2022]
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Metabolic targeting of lactate efflux by malignant glioma inhibits invasiveness and induces necrosis: an in vivo study. Neoplasia 2011; 13:620-32. [PMID: 21750656 DOI: 10.1593/neo.11134] [Citation(s) in RCA: 144] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2011] [Revised: 05/06/2011] [Accepted: 05/12/2011] [Indexed: 11/18/2022] Open
Abstract
Glioblastoma multiforme (GBM) are the most malignant among brain tumors. They are frequently refractory to chemotherapy and radiotherapy with mean patient survival of approximately 6 months, despite surgical intervention. The highly glycolytic nature of glioblastomas describes their propensity to metabolize glucose to lactic acid at an elevated rate. To survive, GBMs efflux lactic acid to the tumor microenvironment through transmembrane transporters denoted monocarboxylate transporters (MCTs). We hypothesized that inhibition of MCT function would impair the glycolytic metabolism and affect both glioma invasiveness and survival. We examined the effect on invasiveness with α-cyano-4-hydroxy-cinnamic acid (ACCA, 4CIN, CHCA), a small-molecule inhibitor of lactate transport, through Matrigel-based and organotypic (brain) slice culture invasive assays using U87-MG and U251-MG glioma cells. We then conducted studies in immunodeficient rats by stereotaxic intracranial implantation of the glioma cells followed by programmed orthotopic application of ACCA through osmotic pumps. Effect on the implanted tumor was monitored by small-animal magnetic resonance imaging. Our assays indicated that glioma invasion was markedly impaired when lactate efflux was inhibited. Convection-enhanced delivery of inhibitor to the tumor bed caused tumor necrosis, with 50% of the animals surviving beyond the experimental end points (3 months after inhibitor exhaustion). Most importantly, control animals did not display any adverse neurologic effects during orthotopic administration of ACCA to brain through programmed delivery. These results indicate the clinical potential of targeting lactate efflux in glioma through delivery of small-molecule inhibitors of MCTs either to the tumor bed or to the postsurgical resection cavity.
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Lee YC, Yoon TJ, Choi GJ, Kim DH. Effect of Triamcinolone on Angiogenesis-related Factors of Cultured Retinal Pigment Epithelial Cells. JOURNAL OF THE KOREAN OPHTHALMOLOGICAL SOCIETY 2009. [DOI: 10.3341/jkos.2009.50.4.594] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Affiliation(s)
- Young Chang Lee
- Department of Ophthalmology, College of Medicine, Chosun University, Gwangju, Korea
| | | | - Gwang Ju Choi
- Department of Ophthalmology, College of Medicine, Chosun University, Gwangju, Korea
| | - Dae Hyun Kim
- Department of Ophthalmology, College of Medicine, Chosun University, Gwangju, Korea
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Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide. Breast Cancer Res 2008; 10:R106. [PMID: 19087284 PMCID: PMC2656903 DOI: 10.1186/bcr2211] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2008] [Revised: 12/08/2008] [Accepted: 12/16/2008] [Indexed: 12/26/2022] Open
Abstract
Introduction At physiologic concentration in serum, the bile acid sodium deoxycholate (DC) induces survival and migration of breast cancer cells. Here we provide evidence of a novel mechanism by which DC reduces apoptosis that is induced by the sphingolipid ceramide in breast cancer cells. Methods Murine mammacarcinoma 4T1 cells were used in vitro to determine apoptosis and alteration of sphingolipid metabolism by DC, and in vivo to quantify the effect of DC on metastasis. Results We found that DC increased the number of intestinal metastases generated from 4T1 cell tumors grafted into the fat pad. The metastatic nodes contained slowly dividing cancer cells in immediate vicinity of newly formed blood vessels. These cells were positive for CD44, a marker that has been suggested to be expressed on breast cancer stem cells. In culture, a subpopulation (3 ± 1%) of slowly dividing, CD44+ cells gave rise to rapidly dividing, CD44- cells. DC promoted survival of CD44+ cells, which was concurrent with reduced levels of activated caspase 3 and ceramide, a sphingolipid inducing apoptosis in 4T1 cells. Z-guggulsterone, an antagonist of the farnesoid-X-receptor, obliterated this anti-apoptotic effect, indicating that DC increased cell survival via farnesoid-X-receptor. DC also increased the gene expression of the vascular endothelial growth factor receptor 2 (Flk-1), suggesting that DC enhanced the initial growth of secondary tumors adjacent to blood vessels. The Flk-1 antagonist SU5416 obliterated the reduction of ceramide and apoptosis by DC, indicating that enhanced cell survival is due to Flk-1-induced reduction in ceramide. Conclusions Our findings show, for the first time, that DC is a natural tumor promoter by elevating Flk-1 and decreasing ceramide-mediated apoptosis of breast cancer progenitor cells. Reducing the level or effect of serum DC and elevating ceramide in breast cancer progenitor cells by treatment with Z-guggulsterone and/or vascular endothelial growth factor receptor 2/Flk-1 antagonists may thus be a promising strategy to reduce breast cancer metastasis.
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An α5β1 integrin inhibitor attenuates glioma growth. Mol Cell Neurosci 2008; 39:579-85. [DOI: 10.1016/j.mcn.2008.08.005] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2008] [Revised: 07/24/2008] [Accepted: 08/13/2008] [Indexed: 11/19/2022] Open
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Meng W, Kallinteri P, Walker DA, Parker TL, Garnett MC. Evaluation of poly (glycerol-adipate) nanoparticle uptake in an in vitro 3-D brain tumor co-culture model. Exp Biol Med (Maywood) 2007; 232:1100-8. [PMID: 17720956 DOI: 10.3181/0612-rm-301] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Despite the inherent problems associated with in vivo animal models of tumor growth and metastases, many of the current in vitro brain tumor models also do not accurately mimic tumor-host brain interactions. Therefore, there is a need to develop such co-culture models to study tumor biology and, importantly, the efficacy of drug delivery systems targeting the brain. So far, few investigations of this nature have been published. In this paper we describe the development of a new model system and its application to drug delivery assessment. For our new model, a co-culture of DAOY cell brain tumor aggregates and organo-typic brain slices was developed. Initially, the DAOY aggregates attached to cerebellum slices and invaded as a unit. Single cells in the periphery of the aggregate detached from the DAOY aggregates and gradually replaced normal brain cells. This invasive behavior of DAOY cells toward organotypic cerebellum slices shows a similar pattern to that seen in vivo. After validation of the co-culture model using transmission electron microscopy, nanoparticle (NP) uptake was then evaluated. Confocal micrographs illustrated that DAOY cells in this co-culture model took up most of the NPs, but few NPs were distributed into brain cells. This finding corresponded with results of NP uptake in DAOY and brain aggregates reported elsewhere.
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Affiliation(s)
- W Meng
- School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, United Kingdom
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Chaichana K, Capilla-Gonzalez V, Gonzalez-Perez O, Pradilla G, Han J, Olivi A, Brem H, Garcia-Verdugo JM, Quiñones-Hinojosa A. Preservation of glial cytoarchitecture from ex vivo human tumor and non-tumor cerebral cortical explants: A human model to study neurological diseases. J Neurosci Methods 2007; 164:261-70. [PMID: 17580092 PMCID: PMC2744592 DOI: 10.1016/j.jneumeth.2007.05.008] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2007] [Revised: 05/04/2007] [Accepted: 05/08/2007] [Indexed: 11/29/2022]
Abstract
For the human brain, in vitro models that accurately represent what occurs in vivo are lacking. Organotypic models may be the closest parallel to human brain tissue outside of a live patient. However, this model has been limited primarily to rodent-derived tissue. We present an organotypic model to maintain intraoperatively collected human tumor and non-tumor explants ex vivo for a prolonged period of time ( approximately 11 days) without any significant changes to the tissue cytoarchitecture as evidenced through immunohistochemistry and electron microscopy analyses. The ability to establish and reliably predict the cytoarchitectural changes that occur with time in an organotypic model of tumor and non-tumor human brain tissue has several potential applications including the study of cell migration on actual tissue matrix, drug toxicity on neural tissue and pharmacological treatment for brain cancers, among others.
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Affiliation(s)
- Kaisorn Chaichana
- Department of Neurosurgery, Cancer Research Building, the Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Vivian Capilla-Gonzalez
- Unidad Mixta Centro de Investigacion Principe Felipe - Universidad de Valencia, Laboratorio de Morfologia Celular, Valencia, Spain
| | - Oscar Gonzalez-Perez
- Laboratory of Neuroscience, School of Psychology, University of Colima, Colima, Mexico
| | - Gustavo Pradilla
- Department of Neurosurgery, Cancer Research Building, the Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - James Han
- Department of Neurosurgery, Cancer Research Building, the Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Alessandro Olivi
- Department of Neurosurgery, Cancer Research Building, the Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Henry Brem
- Department of Neurosurgery, Cancer Research Building, the Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Jose Manuel Garcia-Verdugo
- Unidad Mixta Centro de Investigacion Principe Felipe - Universidad de Valencia, Laboratorio de Morfologia Celular, Valencia, Spain
| | - Alfredo Quiñones-Hinojosa
- Department of Neurosurgery, Cancer Research Building, the Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Jeong YI, Kim SH, Jung TY, Kim IY, Kang SS, Jin YH, Ryu HH, Sun HS, Jin S, Kim KK, Ahn KY, Jung S. Polyion Complex Micelles Composed of All-TranS Retinoic Acid and Poly (Ethylene Glycol)-Grafted-Chitosan. J Pharm Sci 2006; 95:2348-60. [PMID: 16886178 DOI: 10.1002/jps.20586] [Citation(s) in RCA: 64] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
The goal of this study is to develop novel types of polyion complex micelles for the drug delivery to brain tumor. Methoxy poly(ethylene glycol) (mPEG)-grafted chitosan (CP) was synthesized in order to make polymeric micelles encapsulating all-trans retinoic acid (ATRA) based on polyion complex formation. Polyion complex micelles were found to have spherical shapes with sizes of about 50 approximately 200 nm. The loading efficiency of micelle was higher than 80% (w/w) for all formulations. 1H nuclear magnetic resonance (NMR) spectra confirmed the formation of polymeric micelles. The CP graft copolymer and ATRA have distinguishing peaks in their 1H NMR spectra. The specific peaks of ATRA disappeared in D2O or DMSO while it appeared at mixtures of D2O/DMSO, indicating that ATRA and chitosan formed ion complex inner-core. In the cell cytotoxicity study using U87MG cells in vitro, polyion complex micelles showed similar cytotoxicity to that of free ATRA. A migration test was performed to investigate the inhibition of tumor cell invasion in vitro. The results suggested that the polyion complex micelles was more effective at inhibiting tumor cell migration than free ATRA.
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Affiliation(s)
- Young-Il Jeong
- Brain Tumor Research Laboratory, The Research Institute of Medical Science, Chonnam National University Medical School, Gwangju 501-746, Republic of Korea
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Song H, Moon A. Glial cell-derived neurotrophic factor (GDNF) promotes low-grade Hs683 glioma cell migration through JNK, ERK-1/2 and p38 MAPK signaling pathways. Neurosci Res 2006; 56:29-38. [PMID: 16814421 DOI: 10.1016/j.neures.2006.04.019] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2005] [Revised: 03/17/2006] [Accepted: 04/10/2006] [Indexed: 10/24/2022]
Abstract
Invasion of tumor cells is the primary cause of therapeutic failure in the treatment of malignant gliomas. In an attempt to investigate the properties of the malignant progression of glioma cells, we examined the correlation between cell migration and glial cell-derived neurotrophic factor (GDNF) secretion of two glioma cell lines which differ in their invasive phenotypes. Here, we show that the high-grade C6 cells are more migrative and secrete more GDNF than the low-grade Hs683 cells. GDNF signaling is more highly activated in C6 cells than in Hs683 cells. Treatment of the Hs683 cells with GDNF significantly increased migration comparable to the C6 cells, revealing the autocrine and/or paracrine effect of GDNF on promotion of the glioma cell migration. We then examined the involvement of mitogen-activated protein kinases (MAPKs) including c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinases (ERKs) and p38 MAPK in Hs683 cell migration induced by GDNF. A prominent activation of JNK, ERKs and p38 MAPK was observed in the GDNF-treated cells. Functional studies showed that the activation of these MAPKs was critical for Hs683 cell migration induced by GDNF. Our findings revealing molecular mechanisms for the promoting effect of GDNF on glioma cell migration may provide an insight into a better understanding to the malignant progression of human gliomas.
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Affiliation(s)
- Hyun Song
- College of Pharmacy, Duksung Women's University, 419 Ssangmun-Dong, Dobong-Gu, Seoul, Republic of Korea
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Abstract
"Suddenly a solitary horseman appeared on the horizon, then another, then another...in a few moments a whole crowd of horsemen swooped down upon him."-Leacock The illusion of solitary metastases is counterintuitive but has generated a sizable literature on the subject. The reality is that there are more metastatic deaths each year than the total number of true long-term survivors of solitary metastases combining all organ sites in the literature of the past century up to the present time. The largest number of solitary metastases survivors had metastases primarily in the lung and/or liver. With innovations in molecular imaging and advances in molecular oncology, the stage is set to detect truly solitary metastases early. Then, aggressive treatment by surgical excision, stereotactic body radiosurgery, targeted chemotherapy, or immunotherapy could eradicate the lesion. A comprehensive review of solitary metastases in a large variety of anatomic sites is presented. A broader staging system is recommended to encompass a solitary metastasis (M1) and oligometastases (M2) as distinct from multiple metastases (M3).
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Affiliation(s)
- Philip Rubin
- Department of Radiation Oncology, James P. Wilmot Cancer Center at the University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
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Holtkamp N, Afanasieva A, Elstner A, van Landeghem FKH, Könneker M, Kuhn SA, Kettenmann H, von Deimling A. Brain slice invasion model reveals genes differentially regulated in glioma invasion. Biochem Biophys Res Commun 2005; 336:1227-33. [PMID: 16171788 DOI: 10.1016/j.bbrc.2005.08.253] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2005] [Accepted: 08/26/2005] [Indexed: 10/25/2022]
Abstract
Invasion of tumor cells into adjacent brain areas is one of the major problems in treatment of glioma patients. To identify genes that might contribute to invasion, fluorescent F98 glioma cells were allowed to invade an organotypic brain slice. Gene expression analysis revealed 5 up-regulated and 14 down-regulated genes in invasive glioma cells as compared to non-invasive glioma cells. Two gene products, ferritin and cyclin B1, were verified in human gliomas by immunohistochemistry. Ferritin exhibited high mRNA levels in migratory F98 cells and also showed higher protein expression in the infiltrating edge of human gliomas. Cyclin B1 with high mRNA expression levels in stationary F98 cells showed marked protein expression in the central portions of gliomas. These findings are compatible with the concept of tumor cells either proliferating or migrating. Our study is the first to apply brain slice cultures for the identification of differentially regulated genes in glioma invasion.
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Affiliation(s)
- Nikola Holtkamp
- Institute of Neuropathology, Charité Universitätsmedizin Berlin, Germany.
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Schichor C, Kerkau S, Visted T, Martini R, Bjerkvig R, Tonn JC, Goldbrunner R. The brain slice chamber, a novel variation of the Boyden Chamber Assay, allows time-dependent quantification of glioma invasion into mammalian brain in vitro. J Neurooncol 2005; 73:9-18. [PMID: 15933811 DOI: 10.1007/s11060-004-3341-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Glioma cell invasion occurs in a complex micromilieu consisting of neural and glial cells, myelinated fiber tracts, blood vessels and extracellular matrix proteins. The present work describes the brain slice chamber (BSC) as a novel experimental model for assessing invasion of glioma cells into adult mammalian white and gray matter on the basis of the well known Boyden chamber system. As a matrix for invasive tumor cells we used freshly prepared brain tissue from adult pigs. The tissue was sectioned into 40 mum slices that were mechanically fixed to a millipore filter. The neural structures and the three-dimensional architecture of the slice was preserved as verified by immunohistochemistry, light- and electron microscopy. Human U-373 and U87 astrocytoma cells stably transfected with green fluorescent protein (GFP) were assessed for their invasiveness into the brain-slices during a 24 h period. Invasion of U-87 GFP cells was quantified at different time intervals by confocal laser scanning microscopy showing more intense invasion into white compared to gray matter. Two cytostatics (vincristin and paclitaxel) which both are known to affect the cytoskeleton, inhibited glioma cell invasion in a dose dependent manner, which makes the presented model system suitable for functional experiments. In conclusion, the BSC represents a valid and rapid experimental model that may be used to describe the invasive behavior of glioma cells within the preserved three-dimensional structure of mammalian brain tissue in vitro.
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Markovic DS, Glass R, Synowitz M, Rooijen NV, Kettenmann H. Microglia stimulate the invasiveness of glioma cells by increasing the activity of metalloprotease-2. J Neuropathol Exp Neurol 2005; 64:754-62. [PMID: 16141784 DOI: 10.1097/01.jnen.0000178445.33972.a9] [Citation(s) in RCA: 236] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Gliomas represent the most frequent type of human brain tumor, and their strong invasiveness is a significant clinical problem. Microglia, the immunocompetent cells of the brain, contribute significantly to the tumor and are potential interaction partners of the glioma cells. We studied the impact of the presence of microglia on tumor cell invasion in cultured brain slices. To selectively deplete microglia, the slices were treated with clodronate-filled liposomes. When glioma cells were injected into slices devoid of endogenous microglia, the invasiveness of the tumors was significantly decreased as compared with controls. Inoculation of exogenous microglia together with glioma cells into cultured brain slices increased the infiltrative behavior of the tumor depending on the microglia/glioma cell ratio. Cell culture experiments revealed that soluble factors released from glioma cells strongly stimulate metalloprotease-2 activity in microglia. In the brain slices inoculated with glioma cells, increased activity of metalloprotease-2 was directly correlated with the abundance of microglia. Our data indicate that glioma cells stimulate microglial cells to increase breakdown of extracellular matrix and thereby promote tumor invasiveness.
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Affiliation(s)
- Darko S Markovic
- Cellular Neuroscience Group, Max Delbrück Center for Molecular Medicine (MDC), Berlin, Germany
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