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.
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.
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.
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.
Chaudhry NS, Shah AH, Ferraro N, Snelling BM, Bregy A, Madhavan K, Komotar RJ. Predictors of long-term survival in patients with glioblastoma multiforme: advancements from the last quarter century. Cancer Investig. 2013;31(5):287–308. https://doi.org/10.3109/07357907.2013.789899. CrossRef
Paw I, Carpenter RC, Watabe K, Debinski W, Lo HW. Mechanisms regulating glioma invasion. Cancer Lett. 2015;362(1):1–7. https://doi.org/10.1016/j.canlet.2015.03.015. CrossRefPubMedPubMedCentral
Xie Q, Mittal S, Berens ME: Targeting adaptive glioblastoma: an overview of proliferation and invasion. Neuro-Oncology 2014, 16(12):1575–1584.
Skau CT, Fischer RS, Gurel P, Thiam HR, Tubbs A, Baird MA, Davidson MW, Piel M, Alushin GM, Nussenzweig A, et al. FMN2 makes perinuclear actin to protect nuclei during confined migration and promote metastasis. Cell. 2016;167(6):1571–85 e1518. https://doi.org/10.1016/j.cell.2016.10.023. CrossRefPubMedPubMedCentral
Lossi L, Alasia S, Salio C, Merighi A. Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS. Prog Neurobiol. 2009;88(4):221–45. https://doi.org/10.1016/j.pneurobio.2009.01.002. CrossRefPubMed
Aaberg-Jessen C, Norregaard A, Christensen K, Pedersen CB, Andersen C, Kristensen BW. Invasion of primary glioma- and cell line-derived spheroids implanted into corticostriatal slice cultures. Int J Clin Exp Patho. 2013;6(4):546–60.
Jung S, Kim HW, Lee JH, Kang SS, Rhu HH, Jeong YI, Yang SY, Chung HY, Bae CS, Choi C, et al. Brain tumor invasion model system using organotypic brain-slice culture as an alternative to in vivo model. J Cancer Res Clin Oncol. 2002;128(9):469–76. https://doi.org/10.1007/s00432-002-0366-x. CrossRefPubMed
Petterson SA, Jakobsen IP, Jensen SS, Aaberg-Jessen C, Nielsen M, Johansen J, Kristensen BW. Implantation of glioblastoma spheroids into organotypic brain slice cultures as a model for investigating effects of irradiation: a proof of concept. Int J Clin Exp Patho. 2016;9(4):4816–23.
Xu WL, Wang Y, Wu J, Li GY. Quantitative analysis of U251MG human glioma cells invasion in organotypic brain slice co-cultures. Eur Rev Med Pharmaco. 2016;20(11):2221–9.
Korff T, Augustin HG. tensional forces in fibrillar extracellular matrices control directional capillary sprouting. J Cell Sci. 1999;112(Pt 19):3249–58. PubMed
Stahl A, Wu X, Wenger A, Klagsbrun M, Kurschat P. Endothelial progenitor cell sprouting in spheroid cultures is resistant to inhibition by osteoblasts: a model for bone replacement grafts. FEBS Lett. 2005;579(24):5338–42. https://doi.org/10.1016/j.febslet.2005.09.005. CrossRefPubMed
Weber H, Claffey J, Hogan M, Pampillon C, Tacke M. Analyses of Titanocenes in the spheroid-based cellular angiogenesis assay. Toxicol in Vitro. 2008;22(2):531–4. https://doi.org/10.1016/j.tiv.2007.09.014. CrossRefPubMed
Humpel C. Organotypic brain slice cultures: a review. Neuroscience. 2015;305:86–98. https://doi.org/10.1016/j.neuroscience.2015.07.086. CrossRefPubMedPubMedCentral
Mseka T, Cramer LP. Actin depolymerization-based force retracts the cell rear in polarizing and migrating cells. Curr Biol. 2011;21(24):2085–91. https://doi.org/10.1016/j.cub.2011.11.006. CrossRefPubMed
Semple BD, Blomgren K, Gimlin K, Ferriero DM, Noble-Haeusslein LJ. Brain development in rodents and humans: identifying benchmarks of maturation and vulnerability to injury across species. Prog Neurobiol. 2013;106-107:1–16. https://doi.org/10.1016/j.pneurobio.2013.04.001. CrossRefPubMed
Chuang HN, Lohaus R, Hanisch UK, Binder C, Dehghani F, Pukrop T. Coculture system with an organotypic brain slice and 3D spheroid of carcinoma cells. J Vis Exp. 2013;(80) https://doi.org/10.3791/50881.
Chadwick EJ, Yang DP, Filbin MG, Mazzola E, Sun Y, Behar O, Pazyra-Murphy MF, Goumnerova L, Ligon KL, Stiles CD, et al. A brain tumor/Organotypic slice co-culture system for studying tumor microenvironment and targeted drug therapies. J Vis Exp. 2015;105:e53304.
- An advanced glioma cell invasion assay based on organotypic brain slice cultures
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