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In vivo subcellular imaging of tumors in mouse models using a fluorophore-conjugated anti-carcinoembryonic antigen antibody in two-photon excitation microscopy.

Koga S, Oshima Y, Honkura N, Iimura T, Kameda K, Sato K, Yoshida M, Yamamoto Y, Watanabe Y, Hikita A, Imamura T - Cancer Sci. (2014)

Bottom Line: To apply these methods to clinical settings, several groups have developed protocols for fluorescence imaging using antibodies against tumor markers conjugated to fluorescent substances.Although these probes have been useful in macroscopic imaging, the specificity and sensitivity of these methods must be improved to enable them to detect micro-lesions in the early phases of cancer, resulting in better treatment outcomes.These results suggest that two-photon excitation microscopy in conjunction with fluorophore-conjugated antibodies could be widely adapted to detection of cancer-specific cell-surface molecules, both in cancer research and in clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine for Pathogenesis, Ehime University Graduate School of Medicine, Ehime, Japan; Department of Gastrointestinal Surgery and Surgical Oncology, Ehime University Graduate School of Medicine, Ehime, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Ehime, Japan.

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Inoculation of human cancer cells into immunodeficient mice and in vivo macroscopic imaging using a fluorescence zoom microscope. (a) Schema of the sites of inoculation of human cancer cells. The cells were inoculated s.c. into the back skin of nude mice at the rostral–ventral site (HT1080-GFP cells), the caudal–ventral site (HT1080-GFP-CEA cells) or the dorsal site (MKN45-GFP cells). (b) Schema of preparation of skin flaps. Seven or eight days after the inoculation, the inoculation sites were exposed by the skin-flap method. (c–e) In vivo macro imaging of tumors. In vivo macro imaging of the tumor masses was performed using a fluorescence zoom microscope 24 h after injection of Alexa Fluor 594-conjugated anti-CEA antibody (50 μg/mouse). Exposure times for the GFP and Alexa Fluor 594 fluorescence images were 30 and 100 ms, respectively. These experiments were repeated three times and similar results were obtained.
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fig02: Inoculation of human cancer cells into immunodeficient mice and in vivo macroscopic imaging using a fluorescence zoom microscope. (a) Schema of the sites of inoculation of human cancer cells. The cells were inoculated s.c. into the back skin of nude mice at the rostral–ventral site (HT1080-GFP cells), the caudal–ventral site (HT1080-GFP-CEA cells) or the dorsal site (MKN45-GFP cells). (b) Schema of preparation of skin flaps. Seven or eight days after the inoculation, the inoculation sites were exposed by the skin-flap method. (c–e) In vivo macro imaging of tumors. In vivo macro imaging of the tumor masses was performed using a fluorescence zoom microscope 24 h after injection of Alexa Fluor 594-conjugated anti-CEA antibody (50 μg/mouse). Exposure times for the GFP and Alexa Fluor 594 fluorescence images were 30 and 100 ms, respectively. These experiments were repeated three times and similar results were obtained.

Mentions: Next, we attempted to visualize tumor masses of HT1080-GFP-CEA and MKN45-GFP cells in vivo, using an Alexa Fluor 594-conjugated anti-CEA antibody. HT1080-GFP, HT1080-GFP-CEA, and MKN45-GFP cells were s.c. implanted into immunodeficient nude mice, and then Alexa Fluor 594-conjugated anti-CEA antibody was injected into tail vein 7 days after inoculation. Twenty-four hours after the injection, fluorescence macroscopic (macro-scale) images were acquired using a fluorescence zoom microscope after the skin flap operation (Fig.2a,b).22 In the macroscopic images, tumor formation could be confirmed at all the inoculation sites in both the bright field (BF) and GFP images. Although the fluorescence signals in the surrounding tissues were high, tumor masses of CEA-expressing cells (MKN45-GFP and HT1080-GFP-CEA) could be clearly detected in the Alexa Fluor 594 image (Fig.2c–e). However, the intensity of the Alexa Fluor 594 fluorescence signal in the tumor mass of CEA-negative cells was not significantly different from that in surrounding tissues. These results suggest that Alexa Fluor 594-conjugated anti-CEA antibody could specifically visualize tumor masses of CEA-expressing human cancer cells in vivo, but the background signals of the surrounding tissues must be distinguished from those of the tumor mass.


In vivo subcellular imaging of tumors in mouse models using a fluorophore-conjugated anti-carcinoembryonic antigen antibody in two-photon excitation microscopy.

Koga S, Oshima Y, Honkura N, Iimura T, Kameda K, Sato K, Yoshida M, Yamamoto Y, Watanabe Y, Hikita A, Imamura T - Cancer Sci. (2014)

Inoculation of human cancer cells into immunodeficient mice and in vivo macroscopic imaging using a fluorescence zoom microscope. (a) Schema of the sites of inoculation of human cancer cells. The cells were inoculated s.c. into the back skin of nude mice at the rostral–ventral site (HT1080-GFP cells), the caudal–ventral site (HT1080-GFP-CEA cells) or the dorsal site (MKN45-GFP cells). (b) Schema of preparation of skin flaps. Seven or eight days after the inoculation, the inoculation sites were exposed by the skin-flap method. (c–e) In vivo macro imaging of tumors. In vivo macro imaging of the tumor masses was performed using a fluorescence zoom microscope 24 h after injection of Alexa Fluor 594-conjugated anti-CEA antibody (50 μg/mouse). Exposure times for the GFP and Alexa Fluor 594 fluorescence images were 30 and 100 ms, respectively. These experiments were repeated three times and similar results were obtained.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4462348&req=5

fig02: Inoculation of human cancer cells into immunodeficient mice and in vivo macroscopic imaging using a fluorescence zoom microscope. (a) Schema of the sites of inoculation of human cancer cells. The cells were inoculated s.c. into the back skin of nude mice at the rostral–ventral site (HT1080-GFP cells), the caudal–ventral site (HT1080-GFP-CEA cells) or the dorsal site (MKN45-GFP cells). (b) Schema of preparation of skin flaps. Seven or eight days after the inoculation, the inoculation sites were exposed by the skin-flap method. (c–e) In vivo macro imaging of tumors. In vivo macro imaging of the tumor masses was performed using a fluorescence zoom microscope 24 h after injection of Alexa Fluor 594-conjugated anti-CEA antibody (50 μg/mouse). Exposure times for the GFP and Alexa Fluor 594 fluorescence images were 30 and 100 ms, respectively. These experiments were repeated three times and similar results were obtained.
Mentions: Next, we attempted to visualize tumor masses of HT1080-GFP-CEA and MKN45-GFP cells in vivo, using an Alexa Fluor 594-conjugated anti-CEA antibody. HT1080-GFP, HT1080-GFP-CEA, and MKN45-GFP cells were s.c. implanted into immunodeficient nude mice, and then Alexa Fluor 594-conjugated anti-CEA antibody was injected into tail vein 7 days after inoculation. Twenty-four hours after the injection, fluorescence macroscopic (macro-scale) images were acquired using a fluorescence zoom microscope after the skin flap operation (Fig.2a,b).22 In the macroscopic images, tumor formation could be confirmed at all the inoculation sites in both the bright field (BF) and GFP images. Although the fluorescence signals in the surrounding tissues were high, tumor masses of CEA-expressing cells (MKN45-GFP and HT1080-GFP-CEA) could be clearly detected in the Alexa Fluor 594 image (Fig.2c–e). However, the intensity of the Alexa Fluor 594 fluorescence signal in the tumor mass of CEA-negative cells was not significantly different from that in surrounding tissues. These results suggest that Alexa Fluor 594-conjugated anti-CEA antibody could specifically visualize tumor masses of CEA-expressing human cancer cells in vivo, but the background signals of the surrounding tissues must be distinguished from those of the tumor mass.

Bottom Line: To apply these methods to clinical settings, several groups have developed protocols for fluorescence imaging using antibodies against tumor markers conjugated to fluorescent substances.Although these probes have been useful in macroscopic imaging, the specificity and sensitivity of these methods must be improved to enable them to detect micro-lesions in the early phases of cancer, resulting in better treatment outcomes.These results suggest that two-photon excitation microscopy in conjunction with fluorophore-conjugated antibodies could be widely adapted to detection of cancer-specific cell-surface molecules, both in cancer research and in clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine for Pathogenesis, Ehime University Graduate School of Medicine, Ehime, Japan; Department of Gastrointestinal Surgery and Surgical Oncology, Ehime University Graduate School of Medicine, Ehime, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Ehime, Japan.

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Related in: MedlinePlus