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Imaging of human glioblastoma cells and their interactions with mesenchymal stem cells in the zebrafish (Danio rerio) embryonic brain

Milos Vittori

Milos Vittori

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Vittori, Milos
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Barbara Breznik

Barbara Breznik

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Breznik, Barbara
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Tajda Gredar

Tajda Gredar

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Gredar, Tajda
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Katja Hrovat

Katja Hrovat

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Hrovat, Katja
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Lilijana Bizjak Mali

Lilijana Bizjak Mali

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Bizjak Mali, Lilijana
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Tamara T Lah

Tamara T Lah

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Lah, Tamara T
  
26 mar 2016
Imaging of human glioblastoma cells and their interactions with mesenchymal stem cells in the zebrafish (Danio rerio) embryonic brain's Cover Image
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Categoría del artículo: Research Article
Publicado en línea: 26 mar 2016
Páginas: 159 - 167
Recibido: 03 dic 2015
Aceptado: 07 feb 2016
DOI: https://doi.org/10.1515/raon-2016-0017
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© 2016 Radiol Oncol
This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
(A) Clearing of zebrafish embryos. Embryos were fixed 3 days after fertilization, exposed to the different clearing agents SeeDB, sRIMS, ScaleA2, and ScaleU2 for 21 days, and imaged regularly. Time courses of changes in relative transparency are shown, which represents the value of integrated density relative to day 0 divided by the embryo area relative to day 0. Differences between treatments are statistically significant in all cases except between ScaleA2 and Scale B2 (on all days) and between ScaleA2 and sRIMS on day 21. Green, SeeDB; purple, sRIMS; dark blue, ScaleA2; red, ScaleU2; light blue, PBS control. Data for the different clearing agents are displaced horizontally for improved clarity. Means ± SE of eight embryos per treatment are shown. (B) Preservation of GFP fluorescence during clearing. Changes in the detected GFP fluorescence intensity of glioblastoma cells implanted in the brain of zebrafish embryos during treatment with different clearing agents of SeeDB, sRIMS, ScaleA2, and ScaleU2, measured over 21 days of the treatment. The integrated density of GFP-expressing cells relative to day 0 was measured. Fluorescence intensity was significantly increased compared to control in the case of ScaleU2 on all days except on day 3, but not in the case of other clearing agents. Means ±SE of 24 embryos per treatment are shown. (C) Fluorescence of U373-GFP cells in the brain of zebrafish embryos. Representative images show embryos treated with the different clearing agents obtained at the beginning of observation (Day 0, left) and after 3 days of clearing (Day 3, right). The appearance of autofluorescence of the yolk (arrow) is evident in the case of SeeDB. Scale bar: 400 μm.
(A) Clearing of zebrafish embryos. Embryos were fixed 3 days after fertilization, exposed to the different clearing agents SeeDB, sRIMS, ScaleA2, and ScaleU2 for 21 days, and imaged regularly. Time courses of changes in relative transparency are shown, which represents the value of integrated density relative to day 0 divided by the embryo area relative to day 0. Differences between treatments are statistically significant in all cases except between ScaleA2 and Scale B2 (on all days) and between ScaleA2 and sRIMS on day 21. Green, SeeDB; purple, sRIMS; dark blue, ScaleA2; red, ScaleU2; light blue, PBS control. Data for the different clearing agents are displaced horizontally for improved clarity. Means ± SE of eight embryos per treatment are shown. (B) Preservation of GFP fluorescence during clearing. Changes in the detected GFP fluorescence intensity of glioblastoma cells implanted in the brain of zebrafish embryos during treatment with different clearing agents of SeeDB, sRIMS, ScaleA2, and ScaleU2, measured over 21 days of the treatment. The integrated density of GFP-expressing cells relative to day 0 was measured. Fluorescence intensity was significantly increased compared to control in the case of ScaleU2 on all days except on day 3, but not in the case of other clearing agents. Means ±SE of 24 embryos per treatment are shown. (C) Fluorescence of U373-GFP cells in the brain of zebrafish embryos. Representative images show embryos treated with the different clearing agents obtained at the beginning of observation (Day 0, left) and after 3 days of clearing (Day 3, right). The appearance of autofluorescence of the yolk (arrow) is evident in the case of SeeDB. Scale bar: 400 μm.
In vivo imaging of glioblastoma cells in the brain of zebrafish embryos. (A) Embryo 3 days after the implantation of U87DsRed cells in the brain (visible as red fluorescence). Compact tumors have formed in the midbrain and for brain. (B) An embryo with implanted U87-DsRed cells 2 days after implantation, with a string of U87-DsRed cells rapidly invading from the tumor in the brain in the posterior direction (arrow). Scale bars: 300 μm (A); 50 μm (B).
In vivo imaging of glioblastoma cells in the brain of zebrafish embryos. (A) Embryo 3 days after the implantation of U87DsRed cells in the brain (visible as red fluorescence). Compact tumors have formed in the midbrain and for brain. (B) An embryo with implanted U87-DsRed cells 2 days after implantation, with a string of U87-DsRed cells rapidly invading from the tumor in the brain in the posterior direction (arrow). Scale bars: 300 μm (A); 50 μm (B).
Visualization of GBM cells in cleared zebrafish embryos, counterstained with methyl green (presented in blue). Embryos with U373-GFP and U87-DsRed cells implanted in the brain were cleared with ScaleU2, counterstained with methyl green, and imaged with confocal microscopy. (A) U87-DsRed cells (arrows) in the brain of a zebrafish embryo 3 days after implantation. (B) An optical section through the tumor in (A), demonstrating that the tumor is a compact mass of U87 cells in the midbrain ventricle. (C) U373-GFP cells (arrows) in the brain of a zebrafish embryo 3 days after implantation. Elongated U373-GFP cells are visible invading from the tumor (asterisk). (D) A U373-GFP cell invading along the central canal of the spinal cord (arrow), in dorsal view. (E) A U373-GFP cell invading along the central canal of the spinal cord (arrow), in lateral view. Scale bars: 70 μm (A, B, C); 50 μm (D, E).
Visualization of GBM cells in cleared zebrafish embryos, counterstained with methyl green (presented in blue). Embryos with U373-GFP and U87-DsRed cells implanted in the brain were cleared with ScaleU2, counterstained with methyl green, and imaged with confocal microscopy. (A) U87-DsRed cells (arrows) in the brain of a zebrafish embryo 3 days after implantation. (B) An optical section through the tumor in (A), demonstrating that the tumor is a compact mass of U87 cells in the midbrain ventricle. (C) U373-GFP cells (arrows) in the brain of a zebrafish embryo 3 days after implantation. Elongated U373-GFP cells are visible invading from the tumor (asterisk). (D) A U373-GFP cell invading along the central canal of the spinal cord (arrow), in dorsal view. (E) A U373-GFP cell invading along the central canal of the spinal cord (arrow), in lateral view. Scale bars: 70 μm (A, B, C); 50 μm (D, E).
Imaging of co-cultures of GBM cells and MSCs in the brain of zebrafish embryos. A mixture of fluorescent-protein-expressing GBM cells and carbocyaninedye-labeled MSCs was implanted into the brain of the zebrafish embryos. Three days after implantation, the embryos were fixed, cleared in ScaleU2 without the addition of Triton X-100, and imaged with confocal microscopy. (A) The head of a zebrafish embryo with a co-culture of U87-DsRed cells (red) and DiO-labeled MSCs (green) implanted in the brain. (B) The head of a zebrafish embryo with a co-culture of U373-GFP cells (green) and DiI-labeled MSCs (red) implanted in the brain. (C) Invasion of DiI-labeled MSCs (red) along the central canal of the spinal cord. (D) Three-dimensional rendering of a mixed mass of U373 cells (green) and MSCs (red) in a brain obtained from a cleared embryo. Nuclei are stained with methyl green (presented in blue). Scale bars: 250 μm (A, B); 100 μm (C); 50 μm (D).
Imaging of co-cultures of GBM cells and MSCs in the brain of zebrafish embryos. A mixture of fluorescent-protein-expressing GBM cells and carbocyaninedye-labeled MSCs was implanted into the brain of the zebrafish embryos. Three days after implantation, the embryos were fixed, cleared in ScaleU2 without the addition of Triton X-100, and imaged with confocal microscopy. (A) The head of a zebrafish embryo with a co-culture of U87-DsRed cells (red) and DiO-labeled MSCs (green) implanted in the brain. (B) The head of a zebrafish embryo with a co-culture of U373-GFP cells (green) and DiI-labeled MSCs (red) implanted in the brain. (C) Invasion of DiI-labeled MSCs (red) along the central canal of the spinal cord. (D) Three-dimensional rendering of a mixed mass of U373 cells (green) and MSCs (red) in a brain obtained from a cleared embryo. Nuclei are stained with methyl green (presented in blue). Scale bars: 250 μm (A, B); 100 μm (C); 50 μm (D).
Fusion between GBM cells and MSCs in the zebrafish brain. A mixture of U373-GFP cells and DiI-labeled MSCs was implanted in the brain of the embryos, which were fixed, cleared and imaged 3 days after implantation of the cells. Two cells (arrows) emit green GFP fluorescence as well as red DiI fluorescence, which strongly indicates that the U373-GFP cells and MSCs have fused after implantation.Nuclei of embryonic tissues labeled with methyl green. (B) Green fluorescent protein fluorescence of U373 cells. (C) Red fluorescence of DiI, used to label the MSCs. (D) Merged image of all of the fluorescent channels. Scale bar: 200 μm.
Fusion between GBM cells and MSCs in the zebrafish brain. A mixture of U373-GFP cells and DiI-labeled MSCs was implanted in the brain of the embryos, which were fixed, cleared and imaged 3 days after implantation of the cells. Two cells (arrows) emit green GFP fluorescence as well as red DiI fluorescence, which strongly indicates that the U373-GFP cells and MSCs have fused after implantation.Nuclei of embryonic tissues labeled with methyl green. (B) Green fluorescent protein fluorescence of U373 cells. (C) Red fluorescence of DiI, used to label the MSCs. (D) Merged image of all of the fluorescent channels. Scale bar: 200 μm.

Compositions of the selected optical clearing agents

Optical clearing agentCompositionReference
SeeDB80% (w/w) fructose, 0.5% (w/w) α-thioglycerol in waterKe et al.34
sRIMS70% (w/v) sorbitol, 0.01% (w/v) sodium azide, 0.1% (w/v) Tween 20 in 0.02 M phosphate buffer (pH 7.5)Yang et al.39
ScaleA24 M urea, 0.1% (w/v) Triton X-100, 10% (w/w) glycerol in waterHama et al.33
ScaleU24 M urea, 0.1% (w/v) Triton X-100, 30% (w/w) glycerol in waterHama et al.33
Idioma:
Inglés
Calendario de la edición:
4 veces al año
Temas de la revista:
Medicina, Medicina Clínica, Medicina Interna, Hematología, oncología, Radiología
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