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Imaging In Mice With Fluorescent Proteins: From Macro To Subcellular

View Article: PubMed Central - PubMed

ABSTRACT

Whole-body imaging with fluorescent proteins has been shown to be a powerful technology with many applications in small animals. Brighter, red-shifted proteins can make whole-body imaging even more sensitive due to reduced absorption by tissues and less scatter. For example, a new protein called Katushka has been isolated that is the brightest known protein with emission at wavelengths longer than 620 nm. This new protein offers potential for noninvasive whole-body macro imaging such as of tumor growth. For subcellular imaging, to observe cytoplasmic and nuclear dynamics in the living mouse, cancer cells were labeled in the nucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. The nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels was imaged as they trafficked by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Cancer cells trafficking in lymphatic vessels was also imaged. To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment as well as drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of GFP-expressing mice transplanted with the dual-color cancer cells. With the dual-color cancer cells and a highly sensitive small animal imaging system, subcellular dynamics can now be observed in live mice in real time. Fluorescent proteins thus enable both macro and micro imaging technology and thereby provide the basis for the new field of in vivo cell biology.

No MeSH data available.


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Imaging tumor-cell shedding in lymphatic vesselsA footpad tumor, formed after injection of HT1080-GFP-RFP cells, was stimulated by 25- or 250-g weight for 10 s each to increase the internal pressure of the tumor. Stimulations were conducted on the same mouse with a minimum 5-min interval. A cylindrical weight with a 10-mm diameter was used for the stimulation. After stimulation, video rate imaging visualized cancer cell trafficking in the lymphatic vessel with the Olympus OV100 system at x100 magnification for 1 min. The numbers of cell fragments, single cells, and emboli shed into the lymphatic vessel were counted by reviewing the saved movie files. The major axis of the maximum-size shed embolus in each experiment was also measured. A, No weight stimulation onto the footpad. There are only a few fragmented cells in the lymph duct. B, After a 10-s stimulation with a 25-g weight on the footpad, single cells as well as cell fragments are observed trafficking in the lymph duct. C and D, After a 10-s stimulation with the 250-g weight on the footpad, more cell emboli, single cells, and fragments were shed in the lymph duct. Dual-color cell was useful to distinguish the cell condition. D, A high magnification image of the embolus is also shown. Bar 200 μm [36].
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f9-sensors-08-01157: Imaging tumor-cell shedding in lymphatic vesselsA footpad tumor, formed after injection of HT1080-GFP-RFP cells, was stimulated by 25- or 250-g weight for 10 s each to increase the internal pressure of the tumor. Stimulations were conducted on the same mouse with a minimum 5-min interval. A cylindrical weight with a 10-mm diameter was used for the stimulation. After stimulation, video rate imaging visualized cancer cell trafficking in the lymphatic vessel with the Olympus OV100 system at x100 magnification for 1 min. The numbers of cell fragments, single cells, and emboli shed into the lymphatic vessel were counted by reviewing the saved movie files. The major axis of the maximum-size shed embolus in each experiment was also measured. A, No weight stimulation onto the footpad. There are only a few fragmented cells in the lymph duct. B, After a 10-s stimulation with a 25-g weight on the footpad, single cells as well as cell fragments are observed trafficking in the lymph duct. C and D, After a 10-s stimulation with the 250-g weight on the footpad, more cell emboli, single cells, and fragments were shed in the lymph duct. Dual-color cell was useful to distinguish the cell condition. D, A high magnification image of the embolus is also shown. Bar 200 μm [36].

Mentions: We have shown real-time imaging of cancer cell trafficking in lymphatic vessels. Cancer cells labeled with both GFP in the nucleus and RFP in the cytoplasm were injected into the inguinal lymph node of nude mice. The labeled cancer cells trafficked through lymphatic vessels where they were imaged via a skin flap in real time at the cellular level until they entered the axillary lymph node. The bright fluorescence of the cancer cells and the real-time microscopic imaging capability of the Olympus OV100 small-animal imaging system enabled imaging of the trafficking cancer cells in the lymphatics. Using this imaging technology, we investigated the role of pressure on tumor-cell shedding into lymphatic vessels. Pressure was generated by placing 25- and 250-g weights for 10 s on the bottom surface of a tumor-bearing footpad. Tumor cell fragments, single cells, and emboli shed from the footpad tumor were easily distinguished with the labeled cells and OV100 imaging system. Increasing pressure on the tumor increased the numbers of shed cells, fragments, and emboli (Figure 9). Pressure also deformed the shed emboli, increasing their maximum major axis. Imaging lymphatic trafficking of cancer cells can reveal critical steps of lymph node metastasis [36].


Imaging In Mice With Fluorescent Proteins: From Macro To Subcellular
Imaging tumor-cell shedding in lymphatic vesselsA footpad tumor, formed after injection of HT1080-GFP-RFP cells, was stimulated by 25- or 250-g weight for 10 s each to increase the internal pressure of the tumor. Stimulations were conducted on the same mouse with a minimum 5-min interval. A cylindrical weight with a 10-mm diameter was used for the stimulation. After stimulation, video rate imaging visualized cancer cell trafficking in the lymphatic vessel with the Olympus OV100 system at x100 magnification for 1 min. The numbers of cell fragments, single cells, and emboli shed into the lymphatic vessel were counted by reviewing the saved movie files. The major axis of the maximum-size shed embolus in each experiment was also measured. A, No weight stimulation onto the footpad. There are only a few fragmented cells in the lymph duct. B, After a 10-s stimulation with a 25-g weight on the footpad, single cells as well as cell fragments are observed trafficking in the lymph duct. C and D, After a 10-s stimulation with the 250-g weight on the footpad, more cell emboli, single cells, and fragments were shed in the lymph duct. Dual-color cell was useful to distinguish the cell condition. D, A high magnification image of the embolus is also shown. Bar 200 μm [36].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3927509&req=5

f9-sensors-08-01157: Imaging tumor-cell shedding in lymphatic vesselsA footpad tumor, formed after injection of HT1080-GFP-RFP cells, was stimulated by 25- or 250-g weight for 10 s each to increase the internal pressure of the tumor. Stimulations were conducted on the same mouse with a minimum 5-min interval. A cylindrical weight with a 10-mm diameter was used for the stimulation. After stimulation, video rate imaging visualized cancer cell trafficking in the lymphatic vessel with the Olympus OV100 system at x100 magnification for 1 min. The numbers of cell fragments, single cells, and emboli shed into the lymphatic vessel were counted by reviewing the saved movie files. The major axis of the maximum-size shed embolus in each experiment was also measured. A, No weight stimulation onto the footpad. There are only a few fragmented cells in the lymph duct. B, After a 10-s stimulation with a 25-g weight on the footpad, single cells as well as cell fragments are observed trafficking in the lymph duct. C and D, After a 10-s stimulation with the 250-g weight on the footpad, more cell emboli, single cells, and fragments were shed in the lymph duct. Dual-color cell was useful to distinguish the cell condition. D, A high magnification image of the embolus is also shown. Bar 200 μm [36].
Mentions: We have shown real-time imaging of cancer cell trafficking in lymphatic vessels. Cancer cells labeled with both GFP in the nucleus and RFP in the cytoplasm were injected into the inguinal lymph node of nude mice. The labeled cancer cells trafficked through lymphatic vessels where they were imaged via a skin flap in real time at the cellular level until they entered the axillary lymph node. The bright fluorescence of the cancer cells and the real-time microscopic imaging capability of the Olympus OV100 small-animal imaging system enabled imaging of the trafficking cancer cells in the lymphatics. Using this imaging technology, we investigated the role of pressure on tumor-cell shedding into lymphatic vessels. Pressure was generated by placing 25- and 250-g weights for 10 s on the bottom surface of a tumor-bearing footpad. Tumor cell fragments, single cells, and emboli shed from the footpad tumor were easily distinguished with the labeled cells and OV100 imaging system. Increasing pressure on the tumor increased the numbers of shed cells, fragments, and emboli (Figure 9). Pressure also deformed the shed emboli, increasing their maximum major axis. Imaging lymphatic trafficking of cancer cells can reveal critical steps of lymph node metastasis [36].

View Article: PubMed Central - PubMed

ABSTRACT

Whole-body imaging with fluorescent proteins has been shown to be a powerful technology with many applications in small animals. Brighter, red-shifted proteins can make whole-body imaging even more sensitive due to reduced absorption by tissues and less scatter. For example, a new protein called Katushka has been isolated that is the brightest known protein with emission at wavelengths longer than 620 nm. This new protein offers potential for noninvasive whole-body macro imaging such as of tumor growth. For subcellular imaging, to observe cytoplasmic and nuclear dynamics in the living mouse, cancer cells were labeled in the nucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. The nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels was imaged as they trafficked by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Cancer cells trafficking in lymphatic vessels was also imaged. To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment as well as drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of GFP-expressing mice transplanted with the dual-color cancer cells. With the dual-color cancer cells and a highly sensitive small animal imaging system, subcellular dynamics can now be observed in live mice in real time. Fluorescent proteins thus enable both macro and micro imaging technology and thereby provide the basis for the new field of in vivo cell biology.

No MeSH data available.


Related in: MedlinePlus