Fig 1.
OBP-401 labels HCT-116 and HT29 colon cancer cells with GFP in vitro.
Human colon cancer cell lines HCT-116 and HT29 expressing RFP (HCT-116-RFP and HT29-RFP, respectively) were seeded in 6-well plates (1 × 105 cells / well). OBP-401 was added at the indicated multiples of infection (MOI) 24 hours after cell seeding. Images were acquired with an FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan). A. Representative images of non-infected HCT-116-RFP and HT29-RFP cells and HCT-116-RFP and HT29-RFP cells 2, 3, and 4 days after infection of OBP-401 at an MOI of 5, 15 and 30. B. Histogram shows the frequency of GFP-expressing HCT-116 and HT-29 cells at indicated days after infection with OBP-401. C. Representative image of HCT-116-RFP and HT29-RFP cells 5 days after infection of OBP-401 at an MOI of 5, 15, and 30.
Fig 2.
OBP-401 labels high-metastatic colon-cancer-cell selected variants.
A. Scheme of selecting colon cancer high-liver-metastasis cells. HCT-116-RFP cells (2 × 106) were injected into the spleen of female arthymic nude mice (5 weeks old). After 3 cycles of transplantation and harvest, high liver-metastatic colon cancer cells were selected and termed HCT-116L3-RFP. Images were acquired with the FV1000 confocal laser scanning microscope. B. HCT-116-RFP and HCT-116L3-RFP cells were seeded in 6-well plates (1 × 105 cells / well). OBP-401 was added at the indicated multiples of infection (MOI) 24 hours after cell seeding. Images were acquired with the FV1000 confocal laser scanning microscope. Representative images of non-infected HCT-116-RFP and HCT-116L3-RFP cells, and HCT-116-RFP and HCT-116L3-RFP cells 3 days after infection of OBP-401 at an MOI of 5, 15 and 30. C. Histogram shows the frequency of GFP-expressing HCT-116-RFP and HCT-116L3-RFP cells at indicated days after infection of OBP-401. Data are shown as average ± SD. N = 5.
Fig 3.
Establishment of solitary liver-metastasis model.
A. Scheme for establishing solitary live metastasis. B. HCT-116L3-RFP cells (2×105), in Matrigel (BD), were inoculated in the subserosa space of the liver of nude mice (5 weeks old). Panels 1, 2. Open abdominal wall. Panel 3. Exteriorizing the liver. Panel 4. Injection of HCT-116L3-RFP cells in the subserosa of the liver. Panel 5. RFP-fluorescence image of injected HCT-116L3-RFP cells in the subserosa of the liver. Panel 6. Merged image of injected HCT-116L3-RFP cells in the subserosa of the liver. C. Time-course imaging of liver metastasis growth. Upper panels; low magnification images. Lower panels; high magnification images. D. Experimental flowchart with endpoints.
Fig 4.
Comparison of OBP-401-based fluorescence-guided surgery with bright light surgery of a solitary liver metastasis.
OBP-401 was injected intra-tumorally at 1 × 108 PFU when tumors reached approximately 100 mm3 (6 mm diameter). A. Representative whole-liver image of non-infected liver metastasis before and after bright-light surgery (BLS). B. Representative whole-liver image of liver metastasis before injection of OBP-401 and before and after OBP-401-based FGS using the Illumatool imaging system [16]. C. Representative whole-tumor image of resected tumor using BLS. D. Representative image of entire resected tumor using OBP-401-FGS. E. Bar graph shows the comparison of the fluorescent area in the surgical bed after BLS or OBP-401-FGS (left). Bar graph shows the comparison of fluorescence intensity in the surgical bed after BLS or OBP-401-FGS (right). Fluorescence intensity and area are calculated with ImageJ software. Data are shown as average ± SD. N = 16.
Fig 5.
OBP-401-FGS of liver metastasis with the DinoLite hand-held fluorescence scope.
A. Representative bright field and fluorescence images of solitary liver metastasis after labeling with OBP-401 (left). Representative bright field and fluorescence images of solitary liver metastasis with OBP-401-FGS (middle). Representative bright field and fluorescence images of resected metastasis labeled with OBP-401 (left). B. Procedure for OBP-401-FGS with the Dino-Lite hand-held fluorescence-scope.
Fig 6.
OBP-401 labeling visualizes residual cancer cells after BLS.
A. Representative whole-liver images of non-infected orthotopic liver metastasis after bright-light surgery (BLS). B. Representative whole-liver images of orthotopic liver metastasis after OBP-401-FGS. C. Representative images of tumor resected by OBP-401-FGS. D. Representative single-cell image of residual tumor resected by OBP-401-FGS. A-C: Images were acquired with the OV100 whole body fluorescence imaging system. D: Images were acquired with the FV1000 confocal laser scanning microscope.
Fig 7.
OBP-401 labeling visualizes satellite metastasis.
OBP-401-labeled liver and satellite metastases were resected using BLS, and then residual cancer cells were resected using FGS. A. Representative low- (left) and high- (right) magnification images of the margin of the liver metastasis after OBP-401 labeling. B. Representative high-magnification images of complete resection beyond the margin after OBP-401-FGS.
Fig 8.
OBP-401-based FGS prolonged over-all survival compared with BLS.
A. Representative images at necropsy of mice treated with BLS. B. Representative images 120 days after OBP-401-FGS. C. Comparison of fluorescent area of locally-recurrent tumors after BLS or OBP-401-FGS (left). Comparison of fluorescence intensity of locally-recurrent tumors after BLS or OBP-401-FGS (right). Fluorescence intensity is calculated with ImageJ software. Data are shown as average ± SD. N = 16. D. Kaplan-Meyer shows over-all survival after BLS or OBP-401-FGS.
Table 1.
Numbers of animals with and without metastatic recurrence.