Whole-body optical imaging of green Whole-body optical imaging of green fluorescent protein-expressing tumors fluorescent protein-expressing tumors

and metastasesand metastases

Meng Yang*, , , Eugene Baranov*, Ping Meng Yang*, , , Eugene Baranov*, Ping Jiang*, Fang-Xian Sun*, Xiao-Ming Li*, Jiang*, Fang-Xian Sun*, Xiao-Ming Li*, Lingna Li*, Satoshi Hasegawa*, , , Michael Lingna Li*, Satoshi Hasegawa*, , , Michael Bouvet , Maraya Al-Tuwaijri*, , Takashi Bouvet , Maraya Al-Tuwaijri*, , Takashi Chishima*, , , Hiroshi Shimada , A. R. Chishima*, , , Hiroshi Shimada , A. R. Moossa , Sheldon Penman§, and Robert Moossa , Sheldon Penman§, and Robert M. Hoffman*, ,M. Hoffman*, ,¶¶

Stable, high-level GFP-expressing murine melanoma Stable, high-level GFP-expressing murine melanoma transductants in vitro transductants in vitro

External images of murine External images of murine melanoma (B16F0-GFP) melanoma (B16F0-GFP) metastasis in brain. Murine metastasis in brain. Murine melanoma metastases in the melanoma metastases in the mouse brain were imaged by mouse brain were imaged by GFP expression under GFP expression under fluorescence microscopy. fluorescence microscopy. Clear images of metastatic Clear images of metastatic lesions in the brain can be lesions in the brain can be visualized through the scalp visualized through the scalp

and skulland skull

External images of B16F0-External images of B16F0-GFP bone metastasis. In the GFP bone metastasis. In the proximal tibia of the left hind proximal tibia of the left hind leg of C57BL/6 mouse (hair leg of C57BL/6 mouse (hair removed). No metastasis can removed). No metastasis can be detected under bright-field be detected under bright-field microscopy (microscopy (AA). Clear, ). Clear, external images of metastatic external images of metastatic lesions of B16F0-GFP in the lesions of B16F0-GFP in the proximal tibia of the intact proximal tibia of the intact mouse were obtained under mouse were obtained under fluorescence microscopy (fluorescence microscopy (BB). ). Time course metastatic Time course metastatic growth of B16F0-GFP in the growth of B16F0-GFP in the proximal tibia of the intact proximal tibia of the intact nude mouse was imaged nude mouse was imaged externally under fluorescence externally under fluorescence

microscopy (microscopy (CC--EE).).

External images of External images of B16F0-GFP colonizing B16F0-GFP colonizing the liver. A metastatic the liver. A metastatic lesion of B16F0-GFP in lesion of B16F0-GFP in the liver growing at a the liver growing at a depth of 0.8 mm after depth of 0.8 mm after portal vein injection was portal vein injection was externally imaged externally imaged through the abdominal through the abdominal wall of the intact nude wall of the intact nude

mousemouse

External and internal images of liver lesions of AC3488-GFP. (External and internal images of liver lesions of AC3488-GFP. (AA) Lateral, whole-body image of ) Lateral, whole-body image of metastatic liver lesions of a GFP-expressing human colon cancer in the left (thick arrow) and metastatic liver lesions of a GFP-expressing human colon cancer in the left (thick arrow) and right lobes (fine arrow) of a live nude mouse at day 21 after surgical orthotopic transplantation. right lobes (fine arrow) of a live nude mouse at day 21 after surgical orthotopic transplantation. ((BB) Cross-section of mouse shown in ) Cross-section of mouse shown in AA corresponding to the level of the external image of the corresponding to the level of the external image of the

tumor in the liver that was acquired (tumor in the liver that was acquired (AA).).

External and internal images External and internal images of bone metastasis of of bone metastasis of AC3488 GFP. External AC3488 GFP. External fluorescent whole-body fluorescent whole-body images compared with direct images compared with direct images of skeletal images of skeletal metastases. (metastases. (AA) External ) External images of tumors in the images of tumors in the skeletal system including the skeletal system including the skull (arrow heads), scapula skull (arrow heads), scapula (thick arrows), spine (fine (thick arrows), spine (fine arrows), and liver metastasis arrows), and liver metastasis (largest arrows) in a dorsal (largest arrows) in a dorsal view of live, view of live,

Table 1.Table 1.   Minimum-sized fluorescent optical tumour   Minimum-sized fluorescent optical tumour

images at increasing depthimages at increasing depth

Visualizing gene expression by whole-Visualizing gene expression by whole-body fluorescence imagingbody fluorescence imaging

Meng Yang*, Eugene Baranov*, A. R. Moossa , Meng Yang*, Eugene Baranov*, A. R. Moossa , Sheldon Penman , and Robert M. Hoffman*, ,Sheldon Penman , and Robert M. Hoffman*, ,§§

* AntiCancer, Inc., 7917 Ostrow Street, San Diego, CA * AntiCancer, Inc., 7917 Ostrow Street, San Diego, CA 92111;  Department of Surgery, University of California, 92111;  Department of Surgery, University of California, 200 West Arbor Drive, San Diego, CA 92103-8220; and 200 West Arbor Drive, San Diego, CA 92103-8220; and  Department of Biology, Massachusetts Institute of  Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307 02139-4307

Contributed by Sheldon Penman, August 14, 2000 Contributed by Sheldon Penman, August 14, 2000

Materials and Methods Materials and Methods Animals.Animals. Four- to 6-week-old both male and female nude/nude, nude/+, Four- to 6-week-old both male and female nude/nude, nude/+,

and C57BL/6 mice were used. All animal studies were conducted in and C57BL/6 mice were used. All animal studies were conducted in accordance with the principles and procedures outlined in the National accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals under assurance Institutes of Health Guide for the Care and Use of Animals under assurance number A3873-1. Mice were fed with autoclaved laboratory rodent diet number A3873-1. Mice were fed with autoclaved laboratory rodent diet (Tecklad LM-485, Western Research Products, Orange, CA). (Tecklad LM-485, Western Research Products, Orange, CA).

DNA Expression Vector.DNA Expression Vector. The adenoviral (vAd) vector The adenoviral (vAd) vector AdCMV5GFPAE1/AE3 [vAd-green fluorescent protein (GFP)] (Quantum, AdCMV5GFPAE1/AE3 [vAd-green fluorescent protein (GFP)] (Quantum, Montreal, Canada) expresses enhanced GFP and the ampicillin resistance Montreal, Canada) expresses enhanced GFP and the ampicillin resistance gene. gene.

Delivery of vAd-GFP to Various Organs.Delivery of vAd-GFP to Various Organs. BrainBrain. The parietal bone of the . The parietal bone of the skull was exposed after an upper midline scalp incision. Twenty microliters skull was exposed after an upper midline scalp incision. Twenty microliters containing 8 × 1010 plaque-forming units (pfu)/ml vAd-GFP per mouse was containing 8 × 1010 plaque-forming units (pfu)/ml vAd-GFP per mouse was injected in the brain by using a 1-ml 27G1/2 latex-free syringe (Becton injected in the brain by using a 1-ml 27G1/2 latex-free syringe (Becton Dickinson). The puncture hole in the skull was plugged with bone wax. The Dickinson). The puncture hole in the skull was plugged with bone wax. The incision in the scalp was closed with a 7-0 surgical suture in one layer. The incision in the scalp was closed with a 7-0 surgical suture in one layer. The animals were kept under isofluorane anesthesia during surgeryanimals were kept under isofluorane anesthesia during surgery

Fig. 1.  Fig. 1.   External and External and internal images of vAd-GFP internal images of vAd-GFP gene expression in various gene expression in various organs.organs. External fluorescent whole- External fluorescent whole-body images were body images were compared with direct compared with direct images of vAd-GFP gene images of vAd-GFP gene expression in various expression in various organs. Series of external organs. Series of external fluorescence images of fluorescence images of vAd-GFP gene expression vAd-GFP gene expression in brain, liver, pancreas, in brain, liver, pancreas, prostate, and tibia, (prostate, and tibia, (AA, , CC, , EE, , GG, and , and II) compared with ) compared with corresponding images of corresponding images of the exposed organs (the exposed organs (BB, , DD, ,

FF, , HH, and , and JJ).).

Fig. 2.  Fig. 2.   External whole-body image of vAd-GFP gene expression in External whole-body image of vAd-GFP gene expression in the brain. An external image of vAd-GFP gene expression in the brain the brain. An external image of vAd-GFP gene expression in the brain

acquired from a nude mouse in the light box 24 h after gene deliveryacquired from a nude mouse in the light box 24 h after gene delivery

from a series of whole-body images determined that vAd-GFP expression could be first from a series of whole-body images determined that vAd-GFP expression could be first visualized by 5 h, 15 min after local introduction of vAd-GFP gene in a nude mouse. visualized by 5 h, 15 min after local introduction of vAd-GFP gene in a nude mouse. Increases in expression could be visualized in 30-min intervals. Increases in expression could be visualized in 30-min intervals.

Expression [I'GFP] could be first visualized in the liver by 7 h, 45 min after introduction of Expression [I'GFP] could be first visualized in the liver by 7 h, 45 min after introduction of vAd-GFP gene in a nude mouse. Increases in expression could be visualized in 30-min vAd-GFP gene in a nude mouse. Increases in expression could be visualized in 30-min

intervalsintervals

An external External whole-body image of vAd-GFP gene expression in the An external External whole-body image of vAd-GFP gene expression in the liver. image of vAd-GFP gene expression acquired from a nude mouse in the liver. image of vAd-GFP gene expression acquired from a nude mouse in the light box 72 h after gene delivery. Lateral, whole-body image of transgene light box 72 h after gene delivery. Lateral, whole-body image of transgene

expression in the liver can be clearly visualized through the abdominal wallexpression in the liver can be clearly visualized through the abdominal wall

T50 only

T50Luc right ear

T50 Luc right and left

T50Luc no ear

C3H/T50 LUC 0.2x10e6 cells Day 22 after inoculation.

No Luc bladder adsv40

Bladder Luc adsv40

overlay

Open ratbio open rat

overlay

Bladder only

Bio bladder

overlay

Rat no bladder

no bladder Overlay adsv40Luc. WISTAR bladder (catheter) 45 Hrs